Microfluidic system for amplifying and detecting polynucleotides in parallel

ABSTRACT

The present technology provides for an apparatus for detecting polynucleotides in samples, particularly from biological samples. The technology more particularly relates to microfluidic systems that carry out PCR on nucleotides of interest within microfluidic channels, and detect those nucleotides. The apparatus includes a microfluidic cartridge that is configured to accept a plurality of samples, and which can carry out PCR on each sample individually, or a group of, or all of the plurality of samples simultaneously.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/914,109, filed Jun. 26, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/787,977, filed Feb. 11, 2020, which is acontinuation of U.S. patent application Ser. No. 14/796,239, filed Jul.10, 2015, which is a continuation of U.S. patent application Ser. No.13/692,929, filed Dec. 3, 2012 and issued as U.S. Pat. No. 9,080,207 onJul. 14, 2015, which is a continuation of U.S. patent application Ser.No. 13/035,725, filed Feb. 25, 2011, issued as U.S. Pat. No. 8,323,900on Dec. 4, 2012, which is a continuation of U.S. patent application Ser.No. 11/985,577, filed Nov. 14, 2007, issued as U.S. Pat. No. 7,998,708on Aug. 16, 2011, which is a continuation-in-part of U.S. patentapplication Ser. No. 11/728,964, filed Mar. 26, 2007, issued as U.S.Pat. No. 9,040,288 on May 26, 2015, which claims the benefit of U.S.Provisional Patent Application No. 60/786,007, filed Mar. 24, 2006, andU.S. Provisional Patent Application No. 60/859,284, filed Nov. 14, 2006.U.S. patent application Ser. No. 11/985,577 claims the benefit of U.S.Provisional Patent Application No. 60/859,284, filed Nov. 14, 2006, andU.S. Provisional Patent Application No. 60/959,437, filed Jul. 13, 2007.The disclosures of U.S. patent application Ser. No. 13/692,929, U.S.patent application Ser. No. 13/035,725, U.S. patent application Ser. No.11/985,577, U.S. patent application Ser. No. 11/728,964, U.S.Provisional Patent Application No. 60/859,284, and U.S. ProvisionalPatent Application No. 60/959,437 are considered part of the disclosureof this application, and are incorporated by reference herein in theirentirety.

TECHNICAL FIELD

The technology described herein generally relates to systems fordetecting polynucleotides in samples, particularly from biologicalsamples. The technology more particularly relates to microfluidicsystems that carry out PCR on nucleotides of interest withinmicrofluidic channels, and detect those nucleotides.

BACKGROUND

The medical diagnostics industry is a critical element of today'shealthcare infrastructure. At present, however, diagnostic analyses nomatter how routine have become a bottleneck in patient care. There areseveral reasons for this. First, many diagnostic analyses can only bedone with highly specialist equipment that is both expensive and onlyoperable by trained clinicians. Such equipment is found in only a fewlocations—often just one in any given urban area. This means that mosthospitals are required to send out samples for analyses to theselocations, thereby incurring shipping costs and transportation delays,and possibly even sample loss. Second, the equipment in question istypically not available ‘on-demand’ but instead runs in batches, therebydelaying the processing time for many samples because they must wait fora machine to fill up before they can be run.

Understanding that sample flow breaks down into several key steps, itwould be desirable to consider ways to automate as many of these aspossible. For example, a biological sample, once extracted from apatient, must be put in a form suitable for a processing regime thattypically involves using PCR to amplify a vector of interest. Onceamplified, the presence of a nucleotide of interest from the sampleneeds to be determined unambiguously. Sample preparation is a processthat is susceptible to automation but is also relatively routinelycarried out in almost any location. By contrast, steps such as PCR andnucleotide detection have customarily only been within the compass ofspecially trained individuals having access to specialist equipment.

There is a need for a method and apparatus of carrying out PCR anddetection on prepared biological samples, and preferably with highthroughput. In particular there is a need for an easy-to-use device thatcan deliver a diagnostic result on several samples in a short time.

The discussion of the background to the technology herein is included toexplain the context of the technology. This is not to be taken as anadmission that any of the material referred to was published, known, orpart of the common general knowledge as at the priority date of any ofthe claims.

Throughout the description and claims of the specification the word“comprise” and variations thereof, such as “comprising” and “comprises”,is not intended to exclude other additives, components, integers orsteps.

SUMMARY

The present technology addresses systems for detecting polynucleotidesin samples, particularly from biological samples. In particular, thetechnology relates to microfluidic systems that carry out PCR onnucleotides of interest within microfluidic channels, and detect thosenucleotides.

An apparatus, comprising: a receiving bay configured to receive amicrofluidic cartridge; at least one heat source thermally coupled tothe cartridge and configured to carry out PCR on a microdroplet ofpolynucleotide-containing sample, in the cartridge; a detectorconfigured to detect presence of one or more polynucleotides in thesample; and a processor coupled to the detector and the heat source,configured to control heating of one or more regions of the microfluidiccartridge.

A method of carrying out PCR on a plurality of polynucleotide-containingsamples, the method comprising: introducing the plurality of samples into a microfluidic cartridge, wherein the cartridge has a plurality ofPCR reaction chambers configured to permit thermal cycling of theplurality of samples independently of one another; moving the pluralityof samples into the respective plurality of PCR reaction chambers; andamplifying polynucleotides contained with the plurality of samples, byapplication of successive heating and cooling cycles to the PCR reactionchambers.

The details of one or more embodiments of the technology are set forthin the accompanying drawings and further description herein. Otherfeatures, objects, and advantages of the technology will be apparentfrom the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary apparatus, a microfluidic cartridge, and aread head, as further described herein;

FIG. 2 shows an exemplary sample-preparation kit;

FIG. 3 shows a schematic diagram of an apparatus;

FIG. 4 shows a cross-section of a pipetting head and a cartridge inposition in a microfluidic apparatus.

FIG. 5 shows introduction of a PCR-ready sample into a cartridge,situated in an instrument;

FIGS. 6A-6E show exemplary embodiments of an apparatus;

FIG. 7 shows an exploded view of an apparatus;

FIG. 8 shows a block diagram of control circuitry;

FIG. 9 shows a plan view of an exemplary multi-lane microfluidiccartridge;

FIG. 10A shows an exemplary multi-lane cartridge;

FIG. 10B shows a portion of an exemplary multi-lane cartridge;

FIGS. 11A-C show exploded view of an exemplary microfluidic cartridge;

FIG. 12 shows an exemplary highly-multiplexed microfluidic cartridge;

FIGS. 13-16 show various aspects of exemplary highly multiplexedmicrofluidic cartridges; and

FIGS. 17A-C show various aspects of a radially configured highlymultiplexed microfluidic cartridge.

FIG. 18 shows an exemplary microfluidic network in a lane of amulti-lane cartridge;

FIGS. 19A-19D show exemplary microfluidic valves;

FIG. 20 shows an exemplary bubble vent;

FIG. 21 shows a cross-section of a microfluidic cartridge, when incontact with a heater substrate;

FIGS. 22A-22C shows various cut-away sections that can be used toimprove cooling rates during PCR thermal cycling;

FIG. 23 shows a plot of temperature against time during a PCR process,as performed on a microfluidic cartridge as described herein;

FIG. 24 shows an assembly process for a cartridge as further describedherein:

FIGS. 25A and 25B show exemplary deposition of wax droplets intomicrofluidic valves;

FIG. 26 shows an exemplary heater unit;

FIGS. 27A and 27B show a plan view of heater circuitry adjacent to a PCRreaction chamber;

FIG. 27C shows thermal images of heater circuitry in operation;

FIG. 28 shows an overlay of an array of heater elements on an exemplarymulti-lane microfluidic cartridge, wherein various microfluidic networksare visible;

FIG. 29 shows a cross-sectional view of an exemplary detector;

FIG. 30 shows a perspective view of a detector in a read-head;

FIG. 31A, 31B shows a cutaway view of an exemplary detector in aread-head;

FIG. 32 shows an exterior view of an exemplary multiplexed read-headwith an array of detectors therein;

FIG. 33 shows a cutaway view of an exemplary multiplexed read-head, asin FIG. 18;

FIG. 34 shows exemplary pre-amplifier circuitry for a fluorescencedetector;

FIG. 35A shows effects of aperturing on fluorescence intensity; FIG. 35Bshows a detector in cross section with an exemplary aperture;

FIG. 36 shows an exemplary layout for electronics and softwarecomponents, as further described herein;

FIG. 37 shows an exemplary apparatus, a microfluidic cartridge, and aread head, as further described herein;

FIGS. 38-39 show positioning of a cartridge in an exemplary apparatus;

FIGS. 40 and 41 show removal of a heater unit from an exemplaryapparatus;

FIGS. 42A and 42B show an exemplary heater unit and heater substrate;

FIGS. 43A and 43B show an exemplary apparatus having a detector mountedin a sliding lid;

FIGS. 44A-44C show a force member;

FIGS. 45A-45D show a force member associated with a detector;

FIG. 46 shows a block diagram of exemplary electronic circuitry inconjunction with a detector as described herein;

Additional figures are illustrated within the examples, and are furtherdescribed therein.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION Overview of Apparatus

The present technology relates to a system and related methods foramplifying, and carrying out diagnostic analyses on, polynucleotides(e.g., a DNA, RNA, mRNA, or rRNA) from biological samples. For example,the system and methods can determine whether a polynucleotide indicativeof the presence of a particular pathogen (such as a bacterium or avirus) can be present. The polynucleotide may be a sample of genomicDNA, or may be a sample of mitochondrial DNA. The nucleotides aretypically provided to the system having been isolated or released fromparticles such as cells in the sample. The system includes a disposablemicrofluidic cartridge containing multiple sample lanes in parallel anda reusable instrument platform (a PCR analyzer apparatus) that canactuate on-cartridge operations, can detect (e.g., by fluorescencedetection) and analyze the products of the PCR amplification in each ofthe lanes separately, in all simultaneously, or in groupssimultaneously, and, optionally, can display the results on a graphicaluser interface.

A system, microfluidic cartridge, heater unit, detector, kit, methods,and associated computer program product, are now further described.

By cartridge is meant a unit that may be disposable, or reusable inwhole or in part, and that is configured to be used in conjunction withsome other apparatus that has been suitably and complementarilyconfigured to receive and operate on (such as deliver energy to) thecartridge.

By microfluidic, as used herein, is meant that volumes of sample, and/orreagent, and/or amplified polynucleotide are from about 0.1 μl to about999 μl, such as from 1-100 μl, or from 2-25 μl. Similarly, as applied toa cartridge, the term microfluidic means that various components andchannels of the cartridge, as further described herein, are configuredto accept, and/or retain, and/or facilitate passage of microfluidicvolumes of sample, reagent, or amplified polynucleotide.

FIG. 1 shows a perspective view of an exemplary apparatus 100 consistentwith those described herein, as well as various components thereof, suchas exemplary cartridge 200 that contains multiple sample lanes, andexemplary read head 300 that contains detection apparatus for readingsignals from cartridge 200. The apparatus 100 of FIG. 1 is able to carryout real-time PCR on a number of samples in cartridge 200simultaneously. Preferably the number of samples is 12 samples, asillustrated with exemplary cartridge 200, though other numbers ofsamples such as 4, 8, 10, 16, 20, 24, 25, 30, 32, 36, 40, and 48 arewithin the scope of the present description. In preferred operation ofthe apparatus, a PCR-ready solution containing the sample, and,optionally, one or more analyte-specific reagents (ASR's) is prepared,as further described elsewhere (see, e.g., U.S. patent applicationpublication 2006-0166233, incorporated herein by reference), prior tointroduction into cartridge 200. An exemplary kit for preparing aPCR-ready sample, for use with the system described herein, the kitcomprising buffers, lysis pellets, and affinity pellets, is shown inFIG. 2.

System Overview

A schematic overview of a system 981 for carrying out analyses describedherein is shown in FIG. 3. The geometric arrangement of the componentsof system 981 shown in FIG. 3, as well as their respectiveconnectivities, is exemplary and not intended to be limiting.

A processor 980, such as a microprocessor, is configured to controlfunctions of various components of the system as shown, and is therebyin communication with each such component. In particular, processor 980is configured to receive data about a sample to be analyzed, e.g., froma sample reader 990, which may be a barcode reader, an optical characterreader, or an RFID scanner (radio frequency tag reader). For example,the sample identifier can be a handheld bar code reader. Processor 980can be configured to accept user instructions from an input 984, wheresuch instructions may include instructions to start analyzing thesample, and choices of operating conditions.

Processor 980 can also be configured to communicate with an optionaldisplay 982, so that, for example, information including but not limitedto the current status of the system, progress of PCR thermocycling, andany warning message in case of malfunction of either system orcartridge, as well as results of analysis, are transmitted to thedisplay. Additionally, processor 980 may transmit one or more questionsto be displayed on display 982 that prompt a user to provide input inresponse thereto. Thus, in certain embodiments, input 984 and display982 are integrated with one another.

Processor 980 can be optionally further configured to transmit resultsof an analysis to an output device such as a printer, a visual display,or a speaker, or a combination thereof, the transmission being eitherdirectly through a directly dedicated printer cable, or wirelessly, orvia a network connection.

Processor 980 is still further optionally connected via a communicationinterface such as a network interface to a computer network 988. Thecommunication interface can be one or more interfaces selected from thegroup consisting of: a serial connection, a parallel connection, awireless network connection and a wired network connection such as anethernet, firewire, cable connection, or one using USB connectivity.Thereby, when the system is suitably addressed on the network, a remoteuser may access the processor and transmit instructions, input data, orretrieve data, such as may be stored in a memory (not shown) associatedwith the processor, or on some other computer-readable medium that is incommunication with the processor. The computer network connection mayalso permit extraction of data to a remote location, such as a personalcomputer, personal digital assistant, or network storage device such ascomputer server or disk farm. The apparatus may further be configured topermit a user to e-mail results of an analysis directly to some otherparty, such as a healthcare provider, or a diagnostic facility, or apatient.

Although not shown in FIG. 3, in various embodiments, input 984 caninclude one or more input devices selected from the group consisting of:a keyboard, a touch-sensitive surface, a microphone, a track-pad, and amouse. A suitable input device may further comprise a reader offormatted electronic media, such as, but not limited to, a flash memorycard, memory stick, USB-stick, CD, or floppy diskette. An input devicemay further comprise a security feature such as a fingerprint reader,retinal scanner, magnetic strip reader, or bar-code reader, for ensuringthat a user of the system is in fact authorized to do so, according to,for example, pre-loaded identifying characteristics of authorized users.An input device may additionally—and simultaneously—function as anoutput device for writing data in connection with sample analysis. Forexample, if an input device is a reader of formatted electronic media,it may also be a writer of such media. Data that may be written to suchmedia by such a device includes, but is not limited to, environmentalinformation, such as temperature or humidity, pertaining to an analysis,as well as a diagnostic result, and identifying data for the sample inquestion.

Additionally, in various embodiments, the apparatus can further comprisea data storage medium configured to receive data from one or more of theprocessor, an input device, and a communication interface, the datastorage medium being one or more media selected from the groupconsisting of: a hard disk drive, an optical disk drive, or one or moreremovable storage media such as a CD-R, CD-RW, USB-drive, and a flashcard.

Processor 980 is further configured to control various aspects of samplediagnosis, as follows in overview, and as further described in detailherein. The system is configured to operate in conjunction with acomplementary cartridge 994, such as a microfluidic cartridge. Thecartridge is itself configured, as further described herein, to receiveone or more samples 996 containing one or more polynucleotides in a formsuitable for amplification and diagnostic analysis. The cartridge hasdedicated regions within which amplification, such as by PCR, of thepolynucleotides is carried out when the cartridge is situated in theapparatus.

The microfluidic cartridge is received by a receiving bay 992 configuredto selectively receive the cartridge. For example, the receiving bay andthe microfluidic cartridge can be complementary in shape so that themicrofluidic cartridge is selectively received in, e.g., a singleorientation. The microfluidic cartridge can have a registration memberthat fits into a complementary feature of the receiving bay. Theregistration member can be, for example, a cut-out on an edge of thecartridge, such as a corner that is cut-off, or one or more notches thatare made on one or more of the sides. By selectively receiving thecartridge, the receiving bay can help a user to place the cartridge sothat the apparatus can properly operate on the cartridge. The receivingbay can also be configured so that various components of the apparatusthat can operate on the microfluidic cartridge (heat sources, detectors,force members, and the like) are positioned to properly operate on themicrofluidic cartridge. In some embodiments, the apparatus can furtherinclude a sensor coupled to the processor, the sensor configured tosense whether the microfluidic cartridge is selectively received.

The receiving bay is in communication with a heater unit 998 that itselfis controlled by processor 980 in such a way that specific regions ofthe cartridge, such as individual sample lanes, are independently andselectively heated at specific times during amplification and analysis.The processor can be configured to control application of heat to theindividual sample lanes, separately, in all simultaneously, or in groupssimultaneously.

The heat source can be, for example, a contact heat source such as aresistive heater or a network of resistive heaters, or a Peltier device,and the like. The contact heat source can be configured to be in directphysical contact with one or more distinct locations of a microfluidiccartridge received in the receiving bay. In various embodiments, eachcontact source heater can be configured to heat a distinct locationhaving an average diameter in 2 dimensions from about 1 millimeter (mm)to about 15 mm (typically about 1 mm to about 10 mm), or a distinctlocation having a surface area of between about 1 mm² about 225 mm²(typically between about 1 mm² and about 100 mm², or in some embodimentsbetween about 5 mm² and about 50 mm²).

In various embodiments, the heat source can be situated in an assemblythat is removable from the apparatus, for example, to permit cleaning orto replace the heater configuration.

In various embodiments, the apparatus can include a compliant layer atthe contact heat source configured to thermally couple the contact heatsource with at least a portion of a microfluidic cartridge received inthe receiving bay. The compliant layer at can have a thickness ofbetween about 0.05 and about 2 millimeters and a Shore hardness ofbetween about 25 and about 100.

In various embodiments, the apparatus can further include one or moreforce members (not shown in FIG. 3) configured to apply force tothermally couple the at least one heat source at least a portion of amicrofluidic cartridge received in the receiving bay.

In various embodiments, the one or more force members are configured toapply force to a plurality of locations in the microfluidic cartridge.The force applied by the one or more force members can result in anaverage pressure at an interface between a portion of the receiving bayand a portion of the microfluidic cartridge of between about 5kilopascals and about 50 kilopascals, for example, the average pressurecan be at least about 7 kilopascals, and still more preferably at leastabout 14 kilopascals. At least one force member can be manuallyoperated. At least one force member can be mechanically coupled to a lidat the receiving bay, whereby operation of the lid operates the forcemember. The application of force is important to ensure consistentthermal contact between the heater wafer and the PCR reactor andmicrovalves in the microfluidic cartridge.

In various embodiments, the apparatus can further include a lid at thereceiving bay, the lid being operable to at least partially excludeambient light from the receiving bay. The lid can be, for example, asliding lid. The lid can include the optical detector. A major face ofthe lid at the optical detector or at the receiving bay can vary fromplanarity by less than about 100 micrometers, for example, less thanabout 25 micrometers. The lid can be configured to be removable from theapparatus. The lid can include a latching member that ensures that thelid is securely closed before amplification reactions are applied to thesamples in the cartridge.

The processor is also configured to receive signals from and control adetector 999 configured to detect a polynucleotide in a sample in one ormore of the individual sample lanes, separately or simultaneously. Theprocessor thereby provides an indication of a diagnosis from thecartridge 994. Diagnosis can be predicated on the presence or absence ofa specific polynucleotide in a particular sample. The diagnosis can betransmitted to the output device 986 and/or the display 982, asdescribed hereinabove.

The detector can be, for example, an optical detector that includes alight source that selectively emits light in an absorption band of afluorescent dye, and a light detector that selectively detects light inan emission band of the fluorescent dye, wherein the fluorescent dyecorresponds to a fluorescent polynucleotide probe or a fragment thereof.Alternatively, for example, the optical detector can include abandpass-filtered diode that selectively emits light in the absorptionband of the fluorescent dye and a bandpass filtered photodiode thatselectively detects light in the emission band of the fluorescent dye;or for example, the optical detector can be configured to independentlydetect a plurality of fluorescent dyes having different fluorescentemission spectra, wherein each fluorescent dye corresponds to afluorescent polynucleotide probe or a fragment thereof; or for example,the optical detector can be configured to independently detect aplurality of fluorescent dyes at a plurality of different locations on amicrofluidic cartridge, wherein each fluorescent dye corresponds to afluorescent polynucleotide probe or a fragment thereof in a differentsample.

A suitable processor 980 can be designed and manufactured according to,respectively, design principles and semiconductor processing methodsknown in the art.

The system in FIG. 3 is configured so that a cartridge with capacity toreceive multiple samples can be acted upon by the system to analyzemultiple samples—or subsets thereof—simultaneously, or to analyze thesamples consecutively. It is also consistent that additional samples canbe added to a cartridge, while previously added samples are beingamplified and analyzed.

The system shown in outline in FIG. 3, as with other exemplaryembodiments described herein, is advantageous at least because it doesnot require locations within the system suitably configured for storageof reagents. Neither does the system, or other exemplary embodimentsherein, require inlet or outlet ports that are configured to receivereagents from, e.g., externally stored containers such as bottles,canisters, or reservoirs. Therefore, the system in FIG. 3 isself-contained and operates in conjunction with a microfluidiccartridge, wherein the cartridge has locations within it configured toreceive mixtures of sample and PCR reagents.

The system of FIG. 3 may be configured to carry out operation in asingle location, such as a laboratory setting, or may be portable sothat it can accompany, e.g., a physician, or other healthcareprofessional, who may visit patients at different locations. The systemis typically provided with a power-cord so that it can accept AC powerfrom a mains supply or generator. An optional transformer (not shown)built into the system, or situated externally between a power socket andthe system, transforms AC input power into a DC output for use by thesystem. The system may also be configured to operate by using one ormore batteries and therefore is also typically equipped with a batteryrecharging system, and various warning devices that alert a user ifbattery power is becoming too low to reliably initiate or complete adiagnostic analysis.

The system of FIG. 3 may further be configured, for multiplexedcartridge analysis. In one such configuration, multiple instances of asystem, as outlined in FIG. 3, are operated in conjunction with oneanother to accept and to process multiple cartridges, where eachcartridge has been loaded with a different sample. Each component shownin FIG. 3 may therefore be present as many times as there arecartridges, though the various components may be configured in a commonhousing.

In still another configuration, a system is configured to accept and toprocess multiple cartridges, but one or more components in FIG. 3 iscommon to multiple cartridges. For example, a single device may beconfigured with multiple cartridge receiving bays, but a commonprocessor and user interface suitably configured to permit concurrent,consecutive, or simultaneous, control of the various cartridges. In suchan embodiment a single detector, for example, can scan across all of themultiple cartridges. It is further possible that such an embodiment,also utilizes a single sample reader, and a single output device.

In still another configuration, a system as shown in FIG. 3 isconfigured to accept a single cartridge, but wherein the singlecartridge is configured to process more than 1, for example, 2, 3, 4, 5,or 6, samples in parallel, and independently of one another.

It is further consistent with the present technology that a cartridgecan be tagged, e.g., with a molecular bar-code indicative of one or moreof the samples, to facilitate sample tracking, and to minimize risk ofsample mix-up. Methods for such tagging are described elsewhere, e.g.,in U.S. patent application Ser. No. 10/360,854, incorporated herein byreference.

In various embodiments, the apparatus can further include an analysisport. The analysis port can be configured to allow an external sampleanalyzer to analyze a sample in the microfluidic cartridge; for example,the analysis port can be a hole or window in the apparatus which canaccept an optical detection probe that can analyze a sample in situ inthe microfluidic cartridge. In some embodiments, the analysis port canbe configured to direct a sample from the microfluidic cartridge to anexternal sample analyzer; for example, the analysis port can include aconduit in fluid communication with the microfluidic cartridge thatdirect a liquid sample to a chromatography apparatus, an opticalspectrometer, a mass spectrometer, or the like.

Apparatus 100 may optionally comprise one or more stabilizing feet thatcause the body of the device to be elevated above a surface on whichsystem 100 is disposed, thereby permitting ventilation underneath system100, and also providing a user with an improved ability to lift system100. There may be 2, 3, 4, 5, or 6, or more feet, depending upon thesize of system 100. Such feet are preferably made of rubber, or plastic,or metal, and in some embodiments may elevate the body of system 100 byfrom about 2 to about 10 mm above a surface on which it is situated. Thestabilizing function can also be provided by one or more runners thatrun along one or more edges—or are inwardly displaced from one or moreedges—of the underside of the apparatus. Such runners can also be usedin conjunction with one or more feet. In another embodiment, a turntablesituated on the underside permits the apparatus to be rotated in ahorizontal or near-horizontal plane when positioned on, e.g., abenchtop, to facilitate access from a number of angles by a user.

FIG. 4 shows a schematic cross-sectional view of a part of an apparatusas described herein, showing input of sample into a cartridge 200 via apipette 10 (such as a disposable pipette) and an inlet 202. Inlet 202 ispreferably configured to receive a pipette or the bottom end of a PCRtube and thereby accept sample for analysis with minimum waste, and withminimum introduction of air. Cartridge 200 is disposed on top of and incontact with a heater substrate 400. Read head 300 is positioned abovecartridge 200 and a cover for optics 310 restricts the amount of ambientlight that can be detected by the read head.

FIG. 5 shows an example of 4-pipette head used for attaching disposablepipette tips, prior to dispensing PCR-ready sample into a cartridge.

Exemplary Systems

FIGS. 6A-6E show exterior perspective views of various configurations ofan exemplary system, as further described herein. FIG. 6A shows aperspective view of a system 2000 for receiving microfluidic cartridge(not shown), and for causing and controlling various processingoperations to be performed a sample introduced into the cartridge. Theelements of system 2000 are not limited to those explicitly shown. Forexample, although not shown, system 2000 may be connected to a hand-heldbar-code reader, as further described herein.

System 2000 comprises a housing 2002, which can-be made of metal, or ahardened plastic. The form of the housing shown in FIG. 6A embodiesstylistic as well as functional features. Other embodiments of thetechnology may appear somewhat differently, in their arrangement of thecomponents, as well as their overall appearance, in terms of smoothnessof lines, and of exterior finish, and texture. System 2000 furthercomprises one or more stabilizing members 2004. Shown in FIG. 6A is astabilizing foot, of which several are normally present, located atvarious regions of the underside of system 2000 so as to provide balanceand support. For example, there may be three, four, five, six, or eightsuch stabilizing feet. The feet may be moulded into and made of the samematerial as housing 2002, or may be made of one or more separatematerials and attached to the underside of system 2000. For example, thefeet may comprise a rubber that makes it hard for system 2000 to slip ona surface on which it is situated, and also protects the surface fromscratches. The stabilizing member of members may take other forms thanfeet, for example, rails, runners, or one or more pads.

System 2000 further comprises a display 2006, which may be a liquidcrystal display, such as active matrix, an OLED, or some other suitableform. It may present images and other information in color or in blackand white. Display 2006 may also be a touch-sensitive display andtherefore may be configured to accept input from a user in response tovarious displayed prompts. Display 2006 may have an anti-reflectivecoating on it to reduce glare and reflections from overhead lights in anlaboratory setting. Display 2006 may also be illuminated from, e.g., aback-light, to facilitate easier viewing in a dark laboratory.

System 2000, as shown in FIG. 6A, also comprises a moveable lid 2010,having a handle 2008. The lid 2010 can slide back and forward. In FIG.6A, the lid is in a forward position, whereby it is “closed”. In FIG.6B, the lid is shown in a back position, wherein the lid is “open” andreveals a receiving bay 2014 that is configured to receive amicrofluidic cartridge. Of course, as one of ordinary skill in the artwould appreciate, the technology described herein is not limited to alid that slides, or one that slides back and forward. Side to sidemovement is also possible, as is a configuration where the lid is “open”when positioned forward in the device. It is also possible that the lidis a hinged lid, or one that is totally removable.

Handle 2008 performs a role of permitting a user to move lid 2010 fromone position to another, and also performs a role of causing pressure tobe forced down on the lid, when in a closed position, so that pressurecan be applied to a cartridge in the receiving bay 2014. In FIG. 6C,handle 2008 is shown in a depressed position, wherein force is therebyapplied to lid 2014, and thus pressure is applied to a cartridgereceived in the receiving bay beneath the lid.

In one embodiment, the handle and lid assembly are also fitted with amechanical sensor that does not permit the handle to be depressed whenthere is no cartridge in the receiving bay. In another embodiment, thehandle and lid assembly are fitted with a mechanical latch that does notpermit the handle to be raised when an analysis is in progress.

A further configuration of system 2000 is shown in FIG. 6D, wherein adoor 2012 is in an open position. Door 2012 is shown in a closedposition in FIGS. 6A-C. The door is an optional component that permits auser to access a heater module 2020, and also a computer-readable mediuminput tray 2022. System 2000 can function without a door that coversheater module 2020 and medium input 2022, but such a door hasconvenience attached to it. Although the door 2012 is shown hinged atthe bottom, it may also be hinged at one of its sides, or at its upperedge. Door 2012 may alternatively be a removable cover, instead of beinghinged. Door 2012, may also be situated at the rear, or side of system2000 for example, if access to the heater module and/or computerreadable medium input is desired on a different face of the system. Itis also consistent with the system herein that the heater module, andthe computer readable medium input are accessed by separate doors on thesame or different sides of the device, and wherein such separate doorsmay be independently hinged or removable.

Heater module 2020 is preferably removable, and is further describedhereinbelow.

Computer readable medium input 2022 may accept one or more of a varietyof media. Shown in FIG. 2D is an exemplary form of input 2022, a CD-Romtray for accepting a CD, DVD, or mini-CD, or mini-DVD, in any of thecommonly used readable, read-writable, and writable formats. Alsoconsistent with the description herein is an input that can acceptanother form of medium, such as a floppy disc, flash memory such asmemory stick, compact flash, smart data-card, or secure-data card, apen-drive, portable USB-drive, zip-disk, and others. Such an input canalso be configured to accept several different forms of media. Such aninput 2022 is in communication with a processor (as described inconnection with FIG. 3, though not shown in FIGS. 6A-E), that can readdata from a computer-readable medium when properly inserted into theinput.

FIG. 6E shows a plan view of a rear of system 2000. Shown are an airvent 2024, or letting surplus heat escape during an analysis. Typically,on the inside of system 2000, and by air vent 2024 and not shown in FIG.6E, is a fan. Other ports shown in FIG. 6E are as follows: a powersocket 2026 for accepting a power cord that will connect system 2000 toa supply of electricity; an ethernet connection 2028 for linking system2000 to a computer network such as a local area network; an phone-jackconnection 2032 for linking system 2000 to a communication network suchas a telephone network; one or more USB ports 2030, for connectingsystem 2000 to one or more peripheral devices such as a printer, or acomputer hard drive; an infra-red port for communicating with, e.g., aremote controller (not shown), to permit a user to control the systemwithout using a touch-screen interface. For example, a user couldremotely issue scheduling commands to system 2000 to cause it to startan analysis at a specific time in the future.

Features shown on the rear of system 2000 may be arranged in anydifferent manner, depending upon an internal configuration of variouscomponents. Additionally, features shown as being on the rear of system2000, may be optionally presented on another face of system 2000,depending on design preference. Shown in FIG. 6E are exemplaryconnections. It would be understood that various other features,including inputs, outputs, sockets, and connections, may be present onthe rear face of system 2000, though not shown, or on other faces ofsystem 2000.

An exploded view of an exemplary embodiment of the apparatus is shown inFIG. 7, particularly showing internal features of apparatus 2000.Apparatus 2000 can comprise a computer readable medium configured withhardware/firmware that can be employed to drive and monitor theoperations on a cartridge used therewith, as well as software tointerpret, communicate and store the results of a diagnostic testperformed on a sample processed in the cartridge. Referring to FIG. 7,typical components of the apparatus 2000 are shown and include, forexample, control electronics 2005, removable heater/sensor module 2020,detector 2009 such as a fluorescent detection module, display screen oroptionally combined display and user interface 2006 (e.g., a medicalgrade touch sensitive liquid crystal display (LCD)). In someembodiments, lid 2010, detector 2009, and handle 2008 can becollectively referred to as slider module 2007. Additional components ofapparatus 2000 may include one or more mechanical fixtures such as frame2019 to hold the various modules (e.g., the heater/sensor module 2020,and/or the slider module 2007) in alignment, and for providingstructural rigidity. Detector module 2009 can be placed in rails tofacilitate opening and placement of cartridge 2060 in the apparatus2000, and to facilitate alignment of the optics upon closing.Heater/sensor module 2020 can be also placed on rails for easy removaland insertion of the assembly.

Embodiments of apparatus 2000 also include software (e.g., forinterfacing with users, conducting analysis and/or analyzing testresults), firmware (e.g., for controlling the hardware during tests onthe cartridge 812), and one or more peripheral communication interfacesshown collectively as 2031 for peripherals (e.g., communication portssuch as USB/Serial/Ethernet to connect to storage such as compact discor hard disk, to connect input devices such as a bar code reader and/ora keyboard, to connect to other computers or storage via a network, andthe like).

Control electronics 840, shown schematically in the block diagram inFIG. 8, can include one or more functions in various embodiments, forexample for, main control 900, multiplexing 902, display control 904,detector control 906, and the like. The main control function may serveas the hub of control electronics 840 in apparatus 2000 and can managecommunication and control of the various electronic functions. The maincontrol function can also support electrical and communicationsinterface 908 with a user or an output device such as a printer 920, aswell as optional diagnostic and safety functions. In conjunction withmain control function 900, multiplexer function 902 can control sensordata 914 and output current 916 to help control heater/sensor module2020. The display control function 904 can control output to and, ifapplicable, interpret input from touch screen LCD 846, which can therebyprovide a graphical interface to the user in certain embodiments. Thedetector function 906 can be implemented in control electronics 840using typical control and processing circuitry to collect, digitize,filter, and/or transmit the data from a detector 2009 such as one ormore fluorescence detection modules.

Microfluidic Cartridge

The present technology comprises a microfluidic cartridge that isconfigured to carry out an amplification, such as by PCR, of one or morepolynucleotides from one or more samples. It is to be understood that,unless specifically made clear to the contrary, where the term PCR isused herein, any variant of PCR including but not limited to real-timeand quantitative, and any other form of polynucleotide amplification isintended to be encompassed. The microfluidic cartridge need not beself-contained and can be designed so that it receives thermal energyfrom one or more heating elements present in an external apparatus withwhich the cartridge is in thermal communication. An exemplary suchapparatus is further described herein; additional embodiments of such asystem are found in U.S. patent application Ser. No. 11/940,310,entitled “Microfluidic Cartridge and Method of Making Same”, and filedon even date herewith, the specification of which is incorporated hereinby reference.

By cartridge is meant a unit that may be disposable, or reusable inwhole or in part, and that is configured to be used in conjunction withsome other apparatus that has been suitably and complementarilyconfigured to receive and operate on (such as deliver energy to) thecartridge.

By microfluidic, as used herein, is meant that volumes of sample, and/orreagent, and/or amplified polynucleotide are from about 0.1 μl to about999 such as from 1-100 μl, or from 2-25 μl. Similarly, as applied to acartridge, the term microfluidic means that various components andchannels of the cartridge, as further described herein, are configuredto accept, and/or retain, and/or facilitate passage of microfluidicvolumes of sample, reagent, or amplified polynucleotide. Certainembodiments herein can also function with nanolitre volumes (in therange of 10-500 nanoliters, such as 100 nanoliters).

One aspect of the present technology relates to a microfluidic cartridgehaving two or more sample lanes arranged so that analyses can be carriedout in two or more of the lanes in parallel, for example simultaneously,and wherein each lane is independently associated with a given sample.

A sample lane is an independently controllable set of elements by whicha sample can be analyzed, according to methods described herein as wellas others known in the art. A sample lane comprises at least a sampleinlet, and a microfluidic network having one or more microfluidiccomponents, as further described herein.

In various embodiments, a sample lane can include a sample inlet port orvalve, and a microfluidic network that comprises, in fluidiccommunication one or more components selected from the group consistingof: at least one thermally actuated valve, a bubble removal vent, atleast one thermally actuated pump, a gate, mixing channel, positioningelement, microreactor, a downstream thermally actuated valve, and a PCRreaction chamber. The sample inlet valve can be configured to accept asample at a pressure differential compared to ambient pressure ofbetween about 70 and 100 kilopascals.

The cartridge can therefore include a plurality of microfluidicnetworks, each network having various components, and each networkconfigured to carry out PCR on a sample in which the presence or absenceof one or more polynucleotides is to be determined.

A multi-lane cartridge is configured to accept a number of samples inseries or in parallel, simultaneously or consecutively, in particularembodiments 12 samples, wherein the samples include at least a firstsample and a second sample, wherein the first sample and the secondsample each contain one or more polynucleotides in a form suitable foramplification. The polynucleotides in question may be the same as, ordifferent from one another, in different samples and hence in differentlanes of the cartridge. The cartridge typically processes each sample byincreasing the concentration of a polynucleotide to be determined and/orby reducing the concentration of inhibitors relative to theconcentration of polynucleotide to be determined.

The multi-lane cartridge comprises at least a first sample lane having afirst microfluidic network and a second lane having a secondmicrofluidic network, wherein each of the first microfluidic network andthe second microfluidic network is as elsewhere described herein, andwherein the first microfluidic network is configured to amplifypolynucleotides in the first sample, and wherein the second microfluidicnetwork is configured to amplify polynucleotides in the second sample.

In various embodiments, the microfluidic network can be configured tocouple heat from an external heat source to a sample mixture comprisingPCR reagent and neutralized polynucleotide sample under thermal cyclingconditions suitable for creating PCR amplicons from the neutralizedpolynucleotide sample.

At least the external heat source may operate under control of acomputer processor, configured to execute computer readable instructionsfor operating one or more components of each sample lane, independentlyof one another, and for receiving signals from a detector that measuresfluorescence from one or more of the PCR reaction chambers.

For example, FIG. 9 shows a plan view of a microfluidic cartridge 100containing twelve independent sample lanes 101 capable of simultaneousor successive processing. The microfluidic network in each lane istypically configured to carry out amplification, such as by PCR, on aPCR-ready sample, such as one containing nucleic acid extracted from asample using other methods as further described herein. A PCR-readysample is thus typically a mixture comprising the PCR reagents and theneutralized polynucleotide sample, suitable for subjecting to thermalcycling conditions that create PCR amplicons from the neutralizedpolynucleotide sample. For example, a PCR-ready sample can include a PCRreagent mixture comprising a polymerase enzyme, a positive controlplasmid, a fluorogenic hybridization probe selective for at least aportion of the plasmid and a plurality of nucleotides, and at least oneprobe that is selective for a polynucleotide sequence. Exemplary probesare further described herein. Typically, the microfluidic network isconfigured to couple heat from an external heat source with the mixturecomprising the PCR reagent and the neutralized polynucleotide sampleunder thermal cycling conditions suitable for creating PCR ampliconsfrom the neutralized polynucleotide sample.

In various embodiments, the PCR reagent mixture can include a positivecontrol plasmid and a plasmid fluorogenic hybridization probe selectivefor at least a portion of the plasmid, and the microfluidic cartridgecan be configured to allow independent optical detection of thefluorogenic hybridization probe and the plasmid fluorogenichybridization probe.

In various embodiments, the microfluidic cartridge can accommodate anegative control polynucleotide, wherein the microfluidic network can beconfigured to independently carry out PCR on each of a neutralizedpolynucleotide sample and a negative control polynucleotide with the PCRreagent mixture under thermal cycling conditions suitable forindependently creating PCR amplicons of the neutralized polynucleotidesample and PCR amplicons of the negative control polynucleotide. Eachlane of a multi-lane cartridge as described herein can perform tworeactions when used in conjunction with two fluorescence detectionsystems per lane. A variety of combinations of reactions can beperformed in the cartridge, such as two sample reactions in one lane, apositive control and a negative control in two other lanes; or a samplereaction and an internal control in one lane and a negative control in aseparate lane.

FIG. 10A shows a perspective view of a portion of an exemplarymicrofluidic cartridge 200 according to the present technology. FIG. 10Bshows a close-up view of a portion of the cartridge 200 of FIG. 10Aillustrating various representative components. The cartridge 200 may bereferred to as a multi-lane PCR cartridge with dedicated sample inlets202. For example sample inlet 202 is configured to accept a liquidtransfer member (not shown) such as a syringe, a pipette, or a PCR tubecontaining a PCR ready sample. More than one inlet 202 is shown in FIGS.10A, 10B, wherein one inlet operates in conjunction with a single samplelane. Various components of microfluidic circuitry in each lane are alsovisible. For example, microvalves 204, and 206, and hydrophobic vents208 for removing air bubbles, are parts of microfluidic circuitry in agiven lane. Also shown is an ultrafast PCR reactor 210, which, asfurther described herein, is a microfluidic channel in a given samplelane that is long enough to permit PCR to amplify polynucleotidespresent in a sample. Above each PCR reactor 210 is a window 212 thatpermits detection of fluorescence from a fluorescent substance in PCRreactor 210 when a detector is situated above window 212. It is to beunderstood that other configurations of windows are possible including,but not limited to, a single window that straddles each PCR reactoracross the width of cartridge 200.

In preferred embodiments, the multi-sample cartridge has a sizesubstantially the same as that of a 96-well plate as is customarily usedin the art. Advantageously, then, such a cartridge may be used withplate handlers used elsewhere in the art.

The sample inlets of adjacent lanes are reasonably spaced apart from oneanother to prevent any contamination of one sample inlet from anothersample when a user introduces a sample into any one cartridge. In anembodiment, the sample inlets are configured so as to prevent subsequentinadvertent introduction of sample into a given lane after a sample hasalready been introduced into that lane. In certain embodiments, themulti-sample cartridge is designed so that a spacing between thecentroids of sample inlets is 9 mm, which is an industry-recognizedstandard. This means that, in certain embodiments the center-to-centerdistance between inlet holes in the cartridge that accept samples fromPCR tubes, as further described herein, is 9 mm. The inlet holes can bemanufactured conical in shape with an appropriate conical angle so thatindustry-standard pipette tips (2 μl, 20 μl, 200 μl, volumes, etc.) fitsnugly therein. The cartridge herein may be adapted to suit other,later-arising, industry standards not otherwise described herein, aswould be understood by one of ordinary skill in the art.

In one embodiment, an exemplary microfluidic cartridge has 12 samplelanes. The inlet ports in this embodiment have a 6 mm spacing, so that,when used in conjunction with an automated sample loader having 4 heads,spaced equidistantly at 18 mm apart, the inlets can be loaded in threebatches of four inlets: e.g., inlets 1, 4, 7, and 10 together, followedby 2, 5, 8, and 11, then finally 3, 6, 9, and 12, wherein the 12 inletsare numbered consecutively from one side of the cartridge to the otheras shown.

A microfluidic cartridge as used herein may be constructed from a numberof layers. Accordingly, one aspect of the present technology relates toa microfluidic cartridge that comprises a first, second, third, fourth,and fifth layers wherein one or more layers define a plurality ofmicrofluidic networks, each network having various components configuredto carry out PCR on a sample in which the presence or absence of one ormore polynucleotides is to be determined. In various embodiments, one ormore such layers are optional.

FIGS. 11A-C show various views of a layer structure of an exemplarymicrofluidic cartridge comprising a number of layers, as furtherdescribed herein. FIG. 11A shows an exploded view; FIG. 11B shows aperspective view; and FIG. 11C shows a cross-sectional view of a samplelane in the exemplary cartridge. Referring to FIGS. 11A-C, an exemplarymicrofluidic cartridge 400 includes first 420, second 422, third 424,fourth 426, and fifth layers in two non-contiguous parts 428, 430 (asshown) that enclose a microfluidic network having various componentsconfigured to process multiple samples in parallel that include one ormore polynucleotides to be determined.

Microfluidic cartridge 400 can be fabricated as desired. The cartridgecan include a microfluidic substrate layer 424, typically injectionmolded out of a plastic, such as a zeonor plastic (cyclic olefinpolymer), having a PCR channel and valve channels on a first side andvent channels and various inlet holes, including wax loading holes andliquid inlet holes, on a second side (disposed toward hydrophobic ventmembrane 426). It is advantageous that all the microfluidic networkdefining structures, such as PCR reactors, valves, inlet holes, and airvents, are defined on the same single substrate 424. This attributefacilitates manufacture and assembly of the cartridge. Additionally, thematerial from which this substrate is formed is rigid or nondeformable,non-venting to air and other gases, and has a low autofluorescence tofacilitate detection of polynucleotides during an amplification reactionperformed in the microfluidic circuitry defined therein. Rigidity isadvantageous because it facilitates effective and uniform contact with aheat unit as further described herein. Use of a non-venting material isalso advantageous because it reduces the likelihood that theconcentration of various species in liquid form will change duringanalysis. Use of a material having low auto-fluorescence is alsoimportant so that background fluorescence does not detract frommeasurement of fluorescence from the analyte of interest.

The cartridge can further include, disposed on top of the substrate 424,an oleophobic/hydrophobic vent membrane layer 426 of a porous material,such as 0.2 to 1.0 micron pore-size membrane of modifiedpolytetrafluorethylene, the membrane being typically between about 25and about 100 microns thick, and configured to cover the vent channelsof microfluidic substrate 424, and attached thereto using, for example,heat bonding.

Typically, the microfluidic cartridge further includes a layer 428, 430of polypropylene or other plastic label with pressure sensitive adhesive(typically between about 50 and 150 microns thick) configured to sealthe wax loading holes of the valves in substrate 424, trap air used forvalve actuation, and serve as a location for operator markings. In FIG.4A, this layer is shown in two separate pieces, 428, 430, though itwould be understood by one of ordinary skill in the art that a singlepiece layer would be appropriate.

In various embodiments, the label is a computer-readable label. Forexample, the label can include a bar code, a radio frequency tag or oneor more computer-readable characters. The label can be formed of amechanically compliant material. For example, the mechanically compliantmaterial of the label can have a thickness of between about 0.05 andabout 2 millimeters and a Shore hardness of between about 25 and about100. The label can be positioned such that it can be read by a sampleidentification verifier as further described herein.

The cartridge can further include a heat sealable laminate layer 422(typically between about 100 and about 125 microns thick) attached tothe bottom surface of the microfluidic substrate 424 using, for example,heat bonding. This layer serves to seal the PCR channels and ventchannels in substrate 424. The cartridge can further include a thermalinterface material layer 420 (typically about 125 microns thick),attached to the bottom of the heat sealable laminate layer using, forexample, pressure sensitive adhesive. The layer 420 can be compressibleand have a higher thermal conductivity than common plastics, therebyserving to transfer heat across the laminate more efficiently.Typically, however, layer 420 is not present.

The application of pressure to contact the cartridge to the heater of aninstrument that receives the cartridge generally assists in achievingbetter thermal contact between the heater and the heat-receivable partsof the cartridge, and also prevents the bottom laminate structure fromexpanding, as would happen if the PCR channel was only partially filledwith liquid and the air entrapped therein would be thermally expandedduring thermocycling.

In use, cartridge 400 is typically thermally associated with an array ofheat sources configured to operate the components (e.g., valves, gates,actuators, and processing region 410) of the device. Exemplary suchheater arrays are further described herein. Additional embodiments ofheater arrays are described in U.S. patent application Ser. No.11/940,315, entitled “Heater Unit for Microfluidic Diagnostic System”and filed on even date herewith, the specification of which isincorporated herein by reference in its entirety. In some embodiments,the heat sources are controlled by a computer processor and actuatedaccording to a desired protocol. Processors configured to operatemicrofluidic devices are described in, e.g., U.S. application Ser. No.09/819,105, filed Mar. 28, 2001, which application is incorporatedherein by reference.

In various embodiments, during transport and storage, the microfluidiccartridge can be further surrounded by a sealed pouch. The microfluidiccartridge can be sealed in the pouch with an inert gas. The microfluidiccartridge can be disposable for example after one or more of its samplelanes have been used.

Highly Multiplexed Embodiments

Embodiments of the cartridge described herein may be constructed thathave high-density microfluidic circuitry on a single cartridge thatthereby permit processing of multiple samples in parallel, or insequence, on a single cartridge. Preferred numbers of such multiplesamples include 20, 24, 36, 40, 48, 50, 60, 64, 72, 80, 84, 96, and 100,but it would be understood that still other numbers are consistent withthe apparatus and cartridge herein, where deemed convenient andpractical.

Accordingly, different configurations of lanes, sample inlets, andassociated heater networks than those explicitly depicted in the FIGS.and examples that can facilitate processing such numbers of samples on asingle cartridge are within the scope of the instant disclosure.Similarly, alternative configurations of detectors and heating elementsfor use in conjunction with such a highly multiplexed cartridge are alsowithin the scope of the description herein.

It is also to be understood that the microfluidic cartridges describedherein are not to be limited to rectangular shapes, but can includecartridges having circular, elliptical, triangular, rhombohedral,square, and other shapes. Such shapes may also be adapted to includesome irregularity, such as a cut-out, to facilitate placement in acomplementary apparatus as further described herein.

In an exemplary embodiment, a highly multiplexed cartridge has 48 samplelanes, and permits independent control of each valve in each lane bysuitably configured heater circuitry, with 2 banks of thermo cyclingprotocols per lane, as shown in FIG. 12. In the embodiment in FIG. 12,the heaters (shown superimposed on the lanes) are arranged in threearrays 502, 504, with 506, and 508. The heaters are themselves disposedwithin one or more substrates. Heater arrays 502, 508 in two separateglass regions only apply heat to valves in the microfluidic networks ineach lane. Because of the low thermal conductivity of glass, theindividual valves may be heated separately from one another. Thispermits samples to be loaded into the cartridge at different times, andpassed to the PCR reaction chambers independently of one another. ThePCR heaters 504,506 are mounted on a silicon substrate—and are notreadily heated individually, but thereby permit batch processing of PCRsamples, where multiple samples from different lanes are amplified bythe same set of heating/cooling cycles. It is preferable for the PCRheaters to be arranged in 2 banks (the heater arrays 506 on the left andright 508 are not in electrical communication with one another), therebypermitting a separate degree of sample control.

FIG. 13 shows a representative 48-sample cartridge 600 compatible withthe heater arrays of FIG. 12, and having a configuration of inlets 602different to that depicted on other cartridges herein. The inletconfiguration is exemplary and has been designed to maximize efficiencyof space usage on the cartridge. The inlet configuration can becompatible with an automatic pipetting machine that has dispensing headssituated at a 9 mm spacing. For example, such a machine having 4 headscan load 4 inlets at once, in 12 discrete steps, for the cartridge ofFIG. 13. Other configurations of inlets though not explicitly describedor depicted are compatible with the technology described herein.

FIG. 14 shows, in close up, an exemplary spacing of valves 702, channels704, and vents 796, in adjacent lanes 708 of a multi-sample microfluidiccartridge for example as shown in FIG. 13.

FIGS. 15 and 16 show close-ups of, respectively, heater arrays 804compatible with, and inlets 902 on, the exemplary cartridge shown inFIG. 14.

FIGS. 17A and 17B show various views of an embodiment of aradially-configured highly-multiplexed cartridge, having a number ofinlets 1002, microfluidic lanes 1004, valves 1005, and PCR reactionchambers 1006. FIG. 17C shows an array of heater elements 1008compatible with the cartridge layout of FIG. 17A.

The various embodiments shown in FIGS. 12-17C are compatible with liquiddispensers, receiving bays, and detectors that are configureddifferently from the other specific examples described herein.

During the design and manufacture of highly multiplexed cartridges,photolithographic processing steps such as etching, holedrilling/photo-chemical drilling/sand-blasting/ion-milling processesshould be optimized to give well defined holes and microchannel pattern.Proper distances between channels should be identified and maintained toobtain good bonding between the microchannel substrate and the heatconducting substrate layer. In particular, it is desirable that minimaldistances are maintained between pairs of adjacent microchannels topromote, reliable bonding of the laminate in between the channels.

The fabrication by injection molding of these complicated microfluidicstructures having multiple channels and multiple inlet holes entailsproper consideration of dimensional repeatability of these structuresover multiple shots from the injection molding master pattern. Properconsideration is also attached to the placement of ejector pins to pushout the structure from the mold without causing warp, bend or stretchingof it. For example, impression of the ejector pins on the microfluidicsubstrate should not sink into the substrate thereby preventingplanarity of the surface of the cartridge. The accurate placement ofvarious inlet holes (such as sample inlet holes, valve inlet holes andvent holes) relative to adjacent microfluidic channels is also importantbecause the presence of these holes can cause knit-lines to form thatmight cause unintended leak from a hole to a microchannel. Highlymultiplexed microfluidic substrates may be fabricated in other materialssuch as glass, silicon.

The size of the substrate relative to the number of holes is also factorduring fabrication because it is easy to make a substrate having just asimple microfluidic network with a few holes (may be fewer than 10holes) and a few microchannels, but making a substrate having over 24,or over 48, or over 72 holes, etc., is more difficult.

Microfluidic Networks

Particular components of exemplary microfluidic networks are furtherdescribed herein.

Channels of a microfluidic network in a lane of cartridge typically haveat least one sub-millimeter cross-sectional dimension. For example,channels of such a network may have a width and/or a depth of about 1 mmor less (e.g., about 750 microns or less, about 500 microns, or less,about 250 microns or less).

FIG. 18 shows a plan view of a representative microfluidic circuit foundin one lane of a multi-lane cartridge such as shown in FIGS. 10A and10B. It would be understood by one skilled in the art that otherconfigurations of microfluidic network would be consistent with thefunction of the cartridges and apparatus described herein. In operationof the cartridge, in sequence, sample is introduced through liquid inlet202, optionally flows into a bubble removal vent channel 208 (whichpermits adventitious air bubbles introduced into the sample duringentry, to escape), and continues along a channel 216. Typically, whenusing a robotic dispenser of liquid sample, the volume is dispensedaccurately enough that formation of bubbles is not a significantproblem, and the presence of vent channel 208 is not necessary. Thus, incertain embodiments, the bubble removal vent channel 208 is not presentand sample flows directly into channel 216. Throughout the operation ofcartridge 200, the fluid is manipulated as a microdroplet (not shown inthe FIGS.). Valves 204 and 206 are initially both open, so that amicrodroplet of sample-containing fluid can be pumped into PCR reactorchannel 210 from inlet hole 202 under influence of force from the sampleinjection operation. Upon initiating of processing, the detector presenton top of the PCR reactor 210 checks for the presence of liquid in thePCR channel, and then valves 204 and 206 are closed to isolate the PCRreaction mix from the outside. In one embodiment, the checking of thepresence of liquid in the PCR channel is by measuring the heat ramprate, such as by one or more temperature sensors in the heating unit. Achannel with liquid absent will heat up faster than one in which, e.g.,a sample, is present.

Both valves 204 and 206 are closed prior to thermocycling to prevent orreduce any evaporation of liquid, bubble generation, or movement offluid from the PCR reactor. End vent 214 is configured to prevent a userfrom introducing an excess amount of liquid into the microfluidiccartridge, as well as playing a role of containing any sample fromspilling over to unintended parts of the cartridge. A user may inputsample volumes as small as an amount to fill the region from the bubbleremoval vent (if present) to the middle of the microreactor, or up tovalve 204 or beyond valve 204. The use of microvalves prevents both lossof liquid or vapor thereby enabling even a partially filled reactor tosuccessfully complete a PCR thermocycling reaction.

The reactor 210 is a microfluidic channel that is heated through aseries of cycles to carry out amplification of nucleotides in thesample, as further described herein, and according to amplificationprotocols known to those of ordinary skill in the art. The inside wallsof the channel in the PCR reactor are typically made very smooth andpolished to a shiny finish (for example, using a polish selected fromSPI A1, SPI A2, SPI A3, SPI B1, or SPI B2) during manufacture. This isin order to minimize any microscopic quantities of air trapped in thesurface of the PCR channel, which would causing bubbling during thethermocycling steps. The presence of bubbles especially in the detectionregion of the PCR channel could also cause a false or inaccurate readingwhile monitoring progress of the PCR. Additionally, the PCR channel canbe made shallow such that the temperature gradient across the depth ofthe channel is minimized.

The region of the cartridge 212 above PCR reactor 210 is a thinned downsection to reduce thermal mass and autofluorescence from plastic in thecartridge. It permits a detector to more reliably monitor progress ofthe reaction and also to detect fluorescence from a probe that binds toa quantity of amplified nucleotide. Exemplary probes are furtherdescribed herein. The region 212 can be made of thinner material thanthe rest of the cartridge so as to permit the PCR channel to be moreresponsive to a heating cycle (for example, to rapidly heat and coolbetween temperatures appropriate for denaturing and annealing steps),and so as to reduce glare, autofluorescence, and undue absorption offluorescence.

After PCR has been carried out on a sample, and presence or absence of apolynucleotide of interest has been determined, it is preferred that theamplified sample remains in the cartridge and that the cartridge iseither used again (if one or more lanes remain unused), or disposed of.Should a user wish to run a post amplification analysis, such as gelelectrophoresis, the user may pierce a hole through the laminate of thecartridge, and recover an amount—typically about 1.5 microliter—of PCRproduct. The user may also place the individual PCR lane on a specialnarrow heated plate, maintained at a temperature to melt the wax in thevalve, and then aspirate the reacted sample from the inlet hole of thatPCR lane.

In various embodiments, the microfluidic network can optionally includeat least one reservoir configured to contain waste.

Table 1 outlines typical volumes, pumping pressures, and operation timesassociated with various components of a microfluidic cartridge describedherein.

TABLE 1 Operation Pumping Pressure Displacement Volume Time of OperationMoving valve wax ~1-2 psi <1 μl 5-15 seconds plugs Operation Pump UsedPump Design Pump Actuation Moving valve wax Thermopneumatic 1 μl oftrapped air Heat trapped air to plugs pump ~70-90 C.Valves

A valve (sometimes referred to herein as a microvalve) is a component incommunication with a channel, such that the valve has a normally openstate allowing material to pass along a channel from a position on oneside of the valve (e.g., upstream of the valve) to a position on theother side of the valve (e.g., downstream of the valve). Upon actuationof the valve, the valve transitions to a closed state that preventsmaterial from passing along the channel from one side of the valve tothe other. For example, in one embodiment, a valve can include a mass ofa thermally responsive substance (TRS) that is relatively immobile at afirst temperature and more mobile at a second temperature. The first andsecond temperatures are insufficiently high to damage materials, such aspolymer layers of a microfluidic cartridge in which the valve issituated. A mass of TRS can be an essentially solid mass or anagglomeration of smaller particles that cooperate to obstruct thepassage when the valve is closed. Examples of TRS's include a eutecticalloy (e.g., a solder), wax (e.g., an olefin), polymers, plastics, andcombinations thereof. The TRS can also be a blend of variety ofmaterials, such as an emulsion of thermoelastic polymer blended with airmicrobubbles (to enable higher thermal expansion, as well as reversibleexpansion and contraction), polymer blended with expancel material(offering higher thermal expansion), polymer blended with heatconducting microspheres (offering faster heat conduction and hence,faster melting profiles), or a polymer blended with magneticmicrospheres (to permit magnetic actuation of the meltedthermoresponsive material).

Generally, for such a valve, the second temperature is less than about90° C. and the first temperature is less than the second temperature(e.g., about 70° C. or less). Typically, a chamber is in gaseouscommunication with the mass of TRS. The valve is in communication with asource of heat that can be selectively applied to the chamber of air andto the TRS. Upon heating gas (e.g., air) in the chamber and heating themass of TRS to the second temperature, gas pressure within the chamberdue to expansion of the volume of gas, forces the mass to move into thechannel, thereby obstructing material from passing therealong.

An exemplary valve is shown in FIG. 19A. The valve of FIG. 19A has twochambers of air 1203, 1205 in contact with, respectively, each of twochannels 1207, 1208 containing TRS. The air chambers also serve asloading ports for TRS during manufacture of the valve, as furtherdescribed herein. In order to make the valve sealing very robust andreliable, the flow channel 1201 (along which, e.g., sample passes) atthe valve junction is made narrow (typically 150 μm wide, and 150 μmdeep or narrower), and the constricted portion of the flow channel ismade at least 0.5 or 1 mm long such that the TRS seals up a long narrowchannel thereby reducing any leakage through the walls of the channel.In the case of a bad seal, there may be leakage of fluid around walls ofchannel, past the TRS, when the valve is in the closed state. In orderto minimize this, the flow channel is narrowed and elongated as much aspossible. In order to accommodate such a length of channel on acartridge where space may be at a premium, the flow channel canincorporate one or more curves 1209 as shown in FIG. 19A. The valveoperates by heating air in the TRS-loading port, which forces the TRSforwards into the flow-channel in a manner so that it does not come backto its original position. In this way, both air and TRS are heatedduring operation.

In various other embodiments, a valve for use with a microfluidicnetwork in a microfluidic cartridge herein can be a bent valve as shownin FIG. 19B. Such a configuration reduces the footprint of the valve andhence reduces cost per part for highly dense microfluidic cartridges. Asingle valve loading hole 1211 is positioned in the center, that servesas an inlet for thermally responsive substance. The leftmost vent 1213can be configured to be an inlet for, e.g., sample, and the rightmostvent 1215 acts as an exit for, e.g., air. This configuration can be usedas a prototype for testing such attributes as valve and channel geometryand materials.

In various other embodiments, a valve for use with a microfluidicnetwork can include a curved valve as shown in FIG. 19C, in order toreduce the effective cross-section of the valve, thereby enablingmanufacture of cheaper dense microfluidic devices. Such a valve canfunction with a single valve loading hole and air chamber 1221 insteadof a pair as shown in FIG. 19A.

Gates

FIG. 19D shows an exemplary gate as may optionally be used in amicrofluidic network herein. A gate can be a component that can have aclosed state that does not allow material to pass along a channel from aposition on one side of the gate to another side of the gate, and anopen state that does allow material to pass along a channel from aposition on one side of the gate to another side of the gate. Actuationof an open gate can transition the gate to a closed state in whichmaterial is not permitted to pass from one side of the gate (e.g.,upstream of the gate) to the other side of the gate (e.g., downstream ofthe gate). Upon actuation, a closed gate can transition to an open statein which material is permitted to pass from one side of the gate (e.g.,upstream of the gate) to the other side of the gate (e.g., downstream ofthe gate).

In various embodiments, a microfluidic network can include a narrow gate380 as shown in FIG. 19D where a gate loading channel 382 used forloading wax from a wax loading hole 384 to a gate junction 386 can benarrower (e.g., approximately 150 μm wide and 100 microns deep). Anupstream channel 388 as well as a downstream channel 390 of the gatejunction 386 can be made wide (e.g., ˜500 μm) and deep (e.g., ˜500 μm)to help ensure the wax stops at the gate junction 386. The amount ofgate material melted and moved out of the gate junction 386 may beminimized for optimal gate 380 opening. As an off-cartridge heater maybe used to melt the thermally responsive substance in gate 380, amisalignment of the heater could cause the wax in the gate loadingchannel 382 to be melted as well. Therefore, narrowing the dimension ofthe loading channel may increase reliability of gate opening. In thecase of excessive amounts of wax melted at the gate junction 386 andgate loading channel 382, the increased cross-sectional area of thedownstream channel 390 adjacent to the gate junction 386 can prevent waxfrom clogging the downstream channel 390 during gate 380 opening. Thedimensions of the upstream channel 388 at the gate junction 386 can bemade similar to the downstream channel 390 to ensure correct wax loadingduring gate fabrication.

In various embodiments, the gate can be configured to minimize theeffective area or footprint of the gate within the network and thus bentgate configurations, although not shown herein are consistent with theforegoing description.

Vents

In various embodiments, the microfluidic network can include at leastone hydrophobic vent in addition to an end vent. A vent is a generaloutlet (hole) that may or may not be covered with a hydrophobicmembrane. An exit hole is an example of a vent that need not be coveredby a membrane.

A hydrophobic vent (e.g., a vent in FIG. 20) is a structure that permitsgas to exit a channel while limiting (e.g., preventing) quantities ofliquid from exiting the channel. Typically, hydrophobic vents include alayer of porous hydrophobic material (e.g., a porous filter such as aporous hydrophobic membrane from GE Osmonics, Minnetonka, Minn.) thatdefines a wall of the channel. As described elsewhere herein,hydrophobic vents can be used to position a microdroplet of sample at adesired location within a microfluidic network.

The hydrophobic vents of the present technology are preferablyconstructed so that the amount of air that escapes through them ismaximized while minimizing the volume of the channel below the ventsurface. Accordingly, it is preferable that the vent is constructed soas to have a hydrophobic membrane 1303 of large surface area and ashallow cross section of the microchannel below the vent surface.

Hydrophobic vents are useful for bubble removal and typically have alength of at least about 2.5 mm (e.g., at least about 5 mm, at leastabout 7.5 mm) along a channel 1305 (see FIG. 13). The length of thehydrophobic vent is typically at least about 5 times (e.g., at leastabout 10 times, at least about 20 times) larger than a depth of thechannel within the hydrophobic vent. For example, in some embodiments,the channel depth within the hydrophobic vent is about 300 microns orless (e.g., about 250 microns or less, about 200 microns or less, about150 microns or less).

The depth of the channel within the hydrophobic vent is typically about75% or less (e.g., about 65% or less, about 60% or less) of the depth ofthe channel upstream 1301 and downstream (not shown) of the hydrophobicvent. For example, in some embodiments the channel depth within thehydrophobic vent is about 150 microns and the channel depth upstream anddownstream of the hydrophobic vent is about 250 microns. Otherdimensions are consistent with the description herein.

A width of the channel within the hydrophobic vent is typically at leastabout 25% wider (e.g., at least about 50% wider) than a width of thechannel upstream from the vent and downstream from the vent. Forexample, in an exemplary embodiment, the width of the channel within thehydrophobic vent is about 400 microns, and the width of the channelupstream and downstream from the vent is about 250 microns. Otherdimensions are consistent with the description herein.

The vent in FIG. 20 is shown in a linear configuration though it wouldbe understood that it need not be so. A bent, kinked, curved, S-shaped,V-shaped, or U-shaped (as in item 208 FIG. 11) vent is also consistentwith the manner of construction and operation described herein.

Use of Cutaways in Cartridge and Substrate to Improve Rate of CoolingDuring PCR Cycling

During a PCR amplification of a nucleotide sample, a number of thermalcycles are carried out. For improved efficiency, the cooling betweeneach application of heat is preferably as rapid as possible. Improvedrate of cooling can be achieved with various modifications to theheating substrate and/or the cartridge, as shown in FIG. 21.

One way to achieve rapid cooling is to cutaway portions of themicrofluidic cartridge substrate, as shown in FIG. 22A. The upper panelof FIG. 22A is a cross-section of an exemplary microfluidic cartridgetaken along the dashed line A-A′ as marked on the lower panel of FIG.22A. PCR reaction chamber 1601, and representative heaters 1603 areshown. Also shown are two cutaway portions, one of which labeled 1601,that are situated alongside the heaters that are positioned along thelong side of the PCR reaction chamber. Cutaway portions such as 1601reduce the thermal mass of the cartridge, and also permit air tocirculate within the cutaway portions. Both of these aspects permit heatto be conducted away quickly from the immediate vicinity of the PCRreaction chamber. Other configurations of cutouts, such as in shape,position, and number, are consistent with the present technology.

Another way to achieve rapid cooling is to cutaway portions of theheater substrate, as shown in FIG. 22B. The lower panel of FIG. 22B is across-section of an exemplary microfluidic cartridge and heatersubstrate taken along the dashed line A-A′ as marked on the upper panelof FIG. 22B. PCR reaction chamber 901, and representative heaters 1003are shown. Also shown are four cutaway portions, one of which labeled1205, that are situated alongside the heaters that are situated alongthe long side of the PCR reaction chamber. Cutaway portions such as 1205reduce the thermal mass of the heater substrate, and also permit air tocirculate within the cutaway portions. Both of these aspects permit heatto be conducted away quickly from the immediate vicinity of the PCRreaction chamber. Four separate cutaway portions are shown in FIG. 22Aso that control circuitry to the various heaters is not disrupted. Otherconfigurations of cutouts, such as in shape, position, and number, areconsistent with the present technology. These cutouts may be created bya method selected from: selective etching using wet etching processes,deep reactive ion etching, selective etching using CO₂ laser orfemtosecond laser (to prevent surface cracks or stress near thesurface), selective mechanical drilling, selective ultrasonic drilling,or selective abrasive particle blasting. Care has to be taken tomaintain mechanically integrity of the heater while reducing as muchmaterial as possible.

FIG. 22C shows a combination of cutouts and use of ambient air coolingto increase the cooling rate during the cooling stage of thermocycling.A substantial amount of cooling happens by convective loss from thebottom surface of the heater surface to ambient air. The driving forcefor this convective loss is the differential in temperatures between theglass surface and the air temperature. By decreasing the ambient airtemperature by use of, for example, a peltier cooler, the rate ofcooling can be increased. The convective heat loss may also be increasedby keeping the air at a velocity higher than zero.

An example of thermal cycling performance in a PCR reaction chamberobtained with a configuration as described herein, is shown in FIG. 23for a protocol that is set to heat up the reaction mixture to 92° C.,and maintain the temperature for 1 second, then cool to 62° C., and stayfor 10 seconds. The cycle time shown is about 29 seconds, with 8 secondsrequired to heat from 62° C. and stabilize at 92° C., and 10 secondsrequired to cool from 92° C., and stabilize at 62° C. To minimize theoverall time required for a PCR effective to produce detectablequantities of amplified material, it is important to minimize the timerequired for each cycle. Cycle times in the range 15-30 s, such as 18-25s, and 20-22 s, are desirable. In general, an average PCR cycle time of25 seconds as well as cycle times as low as 20 seconds are typical withthe technology described herein. Using reaction volumes less than amicroliter (such as a few hundred nanoliters or less) permits use of anassociated smaller PCR chamber, and enables cycle times as low as 15seconds. An average cycle time of 25 seconds and as low as 20 secondscan be achieved by technology described herein, even without any forcedcooling or implementing any thermal mass reductions described elsewhereherein.

Manufacturing Process for Cartridge

FIG. 24 shows a flow-chart 1800 for an embodiment of an assembly processfor an exemplary cartridge as shown in FIG. 11A herein. It would beunderstood by one of ordinary skill in the art, both that various stepsmay be performed in a different order from the order set forth in FIG.24, and additionally that any given step may be carried out byalternative methods to those described in the figure. It would also beunderstood that, where separate serial steps are illustrated forcarrying out two or more functions, such functions may be performedsynchronously and combined into single steps and remain consistent withthe overall process described herein.

At 1802, a laminate layer is applied to a microfluidic substrate thathas previously been engineered, for example by injection molding, tohave a microfluidic network constructed in it; edges are trimmed fromthe laminate where they spill over the bounds of the substrate.

At 1804, wax is dispensed and loaded into the microvalves of themicrofluidic network in the microfluidic substrate. An exemplary processfor carrying this out is further described herein.

At 1806, the substrate is inspected to ensure that wax from step 1804 isloaded properly and that the laminate from step 1802 adheres properly toit. If a substrate does not satisfy either or both of these tests, it isusually discarded. If substrates repeatedly fail either or both of thesetests, then the wax dispensing, or laminate application steps, asapplicable, are reviewed.

At 1808, a hydrophobic vent membrane is applied to, and heat bonded to,the top of the microfluidic substrate covering at least the one or morevent holes, and on the opposite face of the substrate from the laminate.Edges of the membrane that are in excess of the boundary of thesubstrate are trimmed.

At 1810, the assembly is inspected to ensure that the hydrophobic ventmembrane is bonded well to the microfluidic substrate withoutheat-clogging the microfluidic channels. If any of the channels isblocked, or if the bond between the membrane and the substrate isimperfect, the assembly is discarded, and, in the case of repeateddiscard events, the foregoing process step 1808 is reviewed.

At 1812, optionally, a thermally conductive pad layer is applied to thebottom laminate of the cartridge.

At 1814, two label strips are applied to the top of the microfluidicsubstrate, one to cover the valves, and a second to protect the ventmembranes. It would be understood that a single label strip may bedevised to fulfill both of these roles.

At 1816, additional labels are printed or applied to show identifyingcharacteristics, such as a barcode #, lot # and expiry date on thecartridge. Preferably one or more of these labels has a space and awritable surface that permits a user to make an identifying annotationon the label, by hand.

Optionally, at 1818, to facilitate transport and delivery to a customer,assembled and labeled cartridges are stacked, and cartridges packed intogroups, such as groups of 25, or groups of 10, or groups of 20, orgroups of 48 or 50. Preferably the packaging is via an inert and/ormoisture-free medium.

Wax Loading in Valves

In general, a valve as shown in, e.g., FIGS. 25A-C, is constructed bydepositing a precisely controlled amount of a TRS (such as wax) into aloading inlet machined in the microfluidic substrate. FIGS. 25A and 25Bshow how a combination of controlled hot drop dispensing into a heatedmicrochannel device of the right dimensions and geometry is used toaccurately load wax into a microchannel of a microfluidic cartridge toform a valve. The top of FIG. 25A shows a plan view of a valve inlet 190and loading channel 1902, connecting to a flow channel 1904. The lowerportions of FIG. 25A show the progression of a dispensed wax droplet1906 (having a volume of 75 nl±15 nl) through the inlet 1901 and intothe loading channel 1902.

To accomplish those steps, a heated dispenser head can be accuratelypositioned over the inlet hole of the micro channel in the microfluidicdevice, and can dispense molten wax drops in volumes as small as 75nanoliters with an accuracy of 20%. A suitable dispenser is also onethat can deposit amounts smaller than 100 nl with a precision of +/−20%.The dispenser should also be capable of heating and maintaining thedispensing temperature of the TRS to be dispensed. For example, it mayhave a reservoir to hold the solution of TRS. It is also desirable thatthe dispense head can have freedom of movement at least in a horizontal(x-y) plane so that it can easily move to various locations of amicrofluidic substrate and dispense volumes of TRS into valve inlets atsuch locations without having to be re-set, repositioned manually, orrecalibrated in between each dispense operation.

The inlet hole of the microfluidic cartridge, or other microchanneldevice, is dimensioned in such a way that the droplet of 75 nl can beaccurately propelled to the bottom of the inlet hole using, for example,compressed air, or in a manner similar to an inkjet printing method. Themicrofluidic cartridge is maintained at a temperature above the meltingpoint of the wax thereby permitting the wax to stay in a molten stateimmediately after it is dispensed. After the drop falls to the bottom ofthe inlet hole 1901, the molten wax is drawn into the narrow channel bycapillary action, as shown in the sequence of views in FIG. 25B. Ashoulder between the inlet hole 1901 and the loading channel canfacilitate motion of the TRS. The volume of the narrow section can bedesigned to be approximately equal to a maximum typical amount that isdispensed into the inlet hole. The narrow section can also be designedso that even though the wax dispensed may vary considerably between aminimum and a maximum shot size, the wax always fills up to, and stopsat, the micro channel junction 1907 because the T-junction provides ahigher cross section than that of the narrow section and thus reducesthe capillary forces.

PCR Reagent Mixtures

In various embodiments, the sample for introduction into a lane of themicrofluidic cartridge can include a PCR reagent mixture comprising apolymerase enzyme and a plurality of nucleotides.

In various embodiments, preparation of a PCR-ready sample for use withan apparatus and cartridge as described herein can include contacting aneutralized polynucleotide sample with a PCR reagent mixture comprisinga polymerase enzyme and a plurality of nucleotides (in some embodiments,the PCR reagent mixture can further include a positive control plasmidand a fluorogenic hybridization probe selective for at least a portionof the plasmid).

The PCR-ready sample can be prepared, for example, using the followingsteps: (1) collect sample in sample collection buffer, (2) transferentire sample to lysis tube, mix, heat, and incubate for seven minutes,(3) place on magnetic rack, allow beads to separate, aspiratesupernatant, (4) add 100 μl of Buffer 1, mix, place on magnetic rack,allow beads to separate, aspirate supernatant, (5) add 10 μl of Buffer2, mix, place in high temperature heat block for 3 minutes, place onmagnetic rack, allow beads to separate, transfer 5 μl supernatant, and(6) Add 5 μl of Buffer 3, transfer 1 to 10 μl of supernatant for PCRamplification and detection.

The PCR reagent mixture can be in the form of one or more lyophilizedpellets and the steps by which the PCR-ready sample is prepared caninvolve reconstituting the PCR pellet by contacting it with liquid tocreate a PCR reagent mixture solution. In yet another embodiment, eachof the PCR lanes may have dried down or lyophilized ASR reagentspreloaded such that the user only needs to input prepared polynucleotidesample into the PCR. In another embodiment, the PCR lanes may have onlythe application-specific probes and primers pre-measured and pre-loaded,and the user inputs a sample mixed with the PCR reagents.

In various embodiments, the PCR-ready sample can include at least oneprobe that can be selective for a polynucleotide sequence, wherein thesteps by which the PCR-ready sample is prepared involve contacting theneutralized polynucleotide sample or a PCR amplicon thereof with theprobe. The probe can be a fluorogenic hybridization probe. Thefluorogenic hybridization probe can include a polynucleotide sequencecoupled to a fluorescent reporter dye and a fluorescence quencher dye.

In various embodiments, the PCR-ready sample further includes a samplebuffer.

In various embodiments, the PCR-ready sample includes at least one probethat is selective for a polynucleotide sequence, e.g., thepolynucleotide sequence that is characteristic of a pathogen selectedfrom the group consisting of gram positive bacteria, gram negativebacteria, yeast, fungi, protozoa, and viruses.

In various embodiments, the PCR reagent mixture can further include apolymerase enzyme, a positive control plasmid and a fluorogenichybridization probe selective for at least a portion of the plasmid.

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of an organism, for example any organismthat employs deoxyribonucleic acid or ribonucleic acid polynucleotides.Thus, the probe can be selective for any organism. Suitable organismsinclude mammals (including humans), birds, reptiles, amphibians, fish,domesticated animals, wild animals, extinct organisms, bacteria, fungi,viruses, plants, and the like. The probe can also be selective forcomponents of organisms that employ their own polynucleotides, forexample mitochondria. In some embodiments, the probe is selective formicroorganisms, for example, organisms used in food production (forexample, yeasts employed in fermented products, molds or bacteriaemployed in cheeses, and the like) or pathogens (e.g., of humans,domesticated or wild mammals, domesticated or wild birds, and the like).In some embodiments, the probe is selective for organisms selected fromthe group consisting of gram positive bacteria, gram negative bacteria,yeast, fungi, protozoa, and viruses.

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of an organism selected from the groupconsisting of Staphylococcus spp., e.g., S. epidermidis, S. aureus,Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistantStaphylococcus; Streptococcus (e.g., α, β or γ-hemolytic, Group A, B, C,D or G) such as S. pyogenes, S. agalactiae; E. faecalis, E. durans, andE. faecium (formerly S. faecalis, S. durans, S. faecium);nonenterococcal group D streptococci, e.g., S. bovis and S. equines;Streptococci viridans, e.g., S. mutans, S. sanguis, S. salivarius, S.mitior, A. milleri, S. constellatus, S. intermedius, and S. anginosus;S. iniae; S. pneumoniae; Neisseria, e.g., N. meningitides, N.gonorrhoeae, saprophytic Neisseria sp; Erysipelothrix, e.g., E.rhusiopathiae; Listeria spp., e.g., L. monocytogenes, rarely L. ivanoviiand L. seeligeri; Bacillus, e.g., B. anthracis, B. cereus, B. subtilis,B. subtilus niger, B. thuringiensis; Nocardia asteroids; Legionella,e.g., L. pneumonophilia, Pneumocystis, e.g., P. carinii;Enterobacteriaceae such as Salmonella, Shigella, Escherichia (e.g., E.coli, E. coli O157:H7); Klebsiella, Enterobacter, Serratia, Proteus,Morganella, Providencia, Yersinia, and the like, e.g., Salmonella, e.g.,S. typhi S. paratyphi A, B (S. schottmuelleri), and C (S. hirschfeldii),S. dublin S. choleraesuis, S. enteritidis, S. typhimurium, S.heidelberg, S. newport, S. infantis, S. agona, S. montevideo, and S.saint-paul; Shigella e.g., subgroups: A, B, C, and D, such as S.flexneri, S. sonnei, S. boydii, S. dysenteriae; Proteus (P. mirabilis,P. vulgaris, and P. myxofaciens), Morganella (M. morganii); Providencia(P. rettgeri, P. alcalifaciens, and P. stuartii); Yersinia, e.g., Y.pestis, Y. enterocolitica, Haemophilus, e.g., H. influenzae, H.parainfluenzae H. aphrophilus, H. ducreyi; Brucella, e.g., B. abortus,B. melitensis, B. suis, B. canis; Francisella, e.g., F. tularensis;Pseudomonas, e.g., P. aeruginosa, P. paucimobilis, P. putida, P.fluorescens, P. acidovorans, Burkholderia (Pseudomonas) pseudomallei,Burkholderia mallei, Burkholderia cepacia and Stenotrophomonasmaltophilia; Campylobacter, e.g., C. fetus, C. jejuni, C. pylori(Helicobacter pylori); Vibrio, e.g., V. cholerae, V. parahaemolyticus,V. mimicus, V. alginolyticus, V. hollisae, V. vulnificus, and thenonagglutinable vibrios; Clostridia, e.g., C. perfringens, C. tetani, C.difficile, C. botulinum; Actinomyces, e.g., A. israelii; Bacteroides,e.g., B. fragilis, B. thetaiotaomicron, B. distasonis, B. vulgatus, B.ovatus, B. caccae, and B. merdae; Prevotella, e.g., P. melaninogenica;genus Fusobacterium; Treponema, e.g. T. pallidum subspecies endemicum,T. pallidum subspecies pertenue, T. carateum, and T. pallidum subspeciespallidum; genus Borrelia, e.g., B. burgdorferi; genus Leptospira;Streptobacillus, e.g., S. moniliformis; Spirillum, e.g., S. minus;Mycobacterium, e.g., M. tuberculosis, M. bovis, M. africanum, M. aviumM. intracellulare, M. kansasii, M. xenopi, M. marinum, M. ulcerans, theM. fortuitum complex (M. fortuitum and M. chelonei), M. leprae, M.asiaticum, M. chelonei subsp. abscessus, M. fallax, M. fortuitum, M.malmoense, M. shimoidei, M. simiae, M. szulgai, M. xenopi; Mycoplasma,e.g., M. hominis, M. orale, M. salivarium, M. fermentans, M. pneumoniae,M. bovis, M. tuberculosis, M. avium, M. leprae; Mycoplasma, e.g., M.genitalium; Ureaplasma, e.g., U. urealyticum; Trichomonas, e.g., T.vaginalis; Cryptococcus, e.g., C. neoformans; Histoplasma, e.g., H.capsulatum; Candida, e.g., C. albicans; Aspergillus sp; Coccidioides,e.g., C. immitis; Blastomyces, e.g. B. dermatitidis; Paracoccidioides,e.g., P. brasiliensis; Penicillium, e.g., P. marneffei; Sporothrix,e.g., S. schenckii; Rhizopus, Rhizomucor, Absidia, and Basidiobolus;diseases caused by Bipolaris, Cladophialophora, Cladosporium,Drechslera, Exophiala, Fonsecaea, Phialophora, Xylohypha, Ochroconis,Rhinocladiella, Scolecobasidium, and Wangiella; Trichosporon, e.g., T.beigelii; Blastoschizomyces, e.g., B. capitatus; Plasmodium, e.g., P.falciparum, P. vivax, P. ovale, and P. malariae; Babesia sp; protozoa ofthe genus Trypanosoma, e.g., T. cruzi; Leishmania, e.g., L. donovani, L.major L. tropica, L. mexicana, L. braziliensis, L. viannia braziliensis;Toxoplasma, e.g., T. gondii; Amoebas of the genera Naegleria orAcanthamoeba; Entamoeba histolytica; Giardia lamblia; genusCryptosporidium, e.g., C. parvum; Isospora belli; Cyclosporacayetanensis; Ascaris lumbricoides; Trichuris trichiura; Ancylostomaduodenale or Necator americanus; Strongyloides stercoralis Toxocara,e.g., T. canis, T. cati; Baylisascaris, e.g., B. procyonis; Trichinella,e.g., T. spiralis; Dracunculus, e.g., D. medinensis; genus Filarioidea;Wuchereria bancrofti; Brugia, e.g., B. malayi, or B. timori; Onchocercavolvulus; Loa boa; Dirofilaria immitis; genus Schistosoma, e.g., S.japonicum, S. mansoni, S. mekongi, S. intercalatum, S. haematobium;Paragonimus, e.g., P. Westermani, P. Skriabini; Clonorchis sinensis;Fasciola hepatica; Opisthorchis sp; Fasciolopsis buski; Diphyllobothriumlatum; Taenia, e.g., T. saginata, T. solium; Echinococcus, e.g., E.granulosus, E. multilocularis; Picornaviruses, rhinoviruses echoviruses,coxsackieviruses, influenza virus; paramyxoviruses, e.g., types 1, 2, 3,and 4; adnoviruses; Herpesviruses, e.g., HSV-1 and HSV-2;varicella-zoster virus; human T-lymphotrophic virus (type I and typeII); Arboviruses and Arenaviruses; Togaviridae, Flaviviridae,Bunyaviridae, Reoviridae; Flavivirus; Hantavirus; Viral encephalitis(alphaviruses [e.g., Venezuelan equine encephalitis, eastern equineencephalitis, western equine encephalitis]); Viral hemorrhagic fevers(filoviruses [e.g., Ebola, Marburg] and arenaviruses [e.g., Lassa,Machupo]); Smallpox (variola); retroviruses e.g., human immunodeficiencyviruses 1 and 2; human papillomavirus [HPV] types 6, 11, 16, 18, 31, 33,and 35.

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of an organisms selected from the groupconsisting of Pseudomonas aeruginosa, Proteus mirabilis, Klebsiellaoxytoca, Klebsiella pneumoniae, Escherichia coli, AcinetobacterBaumannii, Serratia marcescens, Enterobacter aerogenes, Enterococcusfaecium, vancomycin-resistant enterococcus (VRE), Staphylococcus aureus,methecillin-resistant Staphylococcus aureus(MRSA), Streptococcusviridans, Listeria monocytogenes, Enterococcus spp., Streptococcus GroupB, Streptococcus Group C, Streptococcus Group G, Streptococcus Group F,Enterococcus faecalis, Streptococcus pneumoniae, Staphylococcusepidermidis, Gardenerella vaginalis, Micrococcus sps., Haemophilusinfluenzae, Neisseria gonorrhoeee, Moraxella catarrahlis, Salmonellasps., Chlamydia trachomatis, Peptostreptococcus productus,Peptostreptococcus anaerobius, Lactobacillus fermentum, Eubacteriumlentum, Candida glabrata, Candida albicans, Chlamydia spp., Camplobacterspp., Salmonella spp., smallpox (variola major), Yersina Pestis, HerpesSimplex Virus I (HSV I), and Herpes Simplex Virus II (HSV II).

In various embodiments, the probe can be selective for a polynucleotidesequence that is characteristic of Group B Streptococcus.

In various embodiments, a method of carrying out PCR on a sample canfurther include one or more of the following steps: heating thebiological sample in the microfluidic cartridge; pressurizing thebiological sample in the microfluidic cartridge at a pressuredifferential compared to ambient pressure of between about 20kilopascals and 200 kilopascals, or in some embodiments, between about70 kilopascals and 110 kilopascals.

In some embodiments, the method for sampling a polynucleotide caninclude the steps of: placing a microfluidic cartridge containing aPCR-ready sample in a receiving bay of a suitably configured apparatus;carrying out PCR on the sample under thermal cycling conditions suitablefor creating PCR amplicons from the neutralized polynucleotide in thesample, the PCR-ready sample comprising a polymerase enzyme, a positivecontrol plasmid, a fluorogenic hybridization probe selective for atleast a portion of the plasmid, and a plurality of nucleotides;contacting the neutralized polynucleotide sample or a PCR ampliconthereof with the at least one fluorogenic probe that is selective for apolynucleotide sequence, wherein the probe is selective for apolynucleotide sequence that is characteristic of an organism selectedfrom the group consisting of gram positive bacteria, gram negativebacteria, yeast, fungi, protozoa, and viruses; and detecting thefluorogenic probe, the presence of the organism for which the onefluorogenic probe is selective is determined.

Carrying out PCR on a PCR-ready sample can additionally include:independently contacting each of the neutralized polynucleotide sampleand a negative control polynucleotide with the PCR reagent mixture underthermal cycling conditions suitable for independently creating PCRamplicons of the neutralized polynucleotide sample and PCR amplicons ofthe negative control polynucleotide; and/or contacting the neutralizedpolynucleotide sample or a PCR amplicon thereof and the negative controlpolynucleotide or a PCR amplicon thereof with at least one probe that isselective for a polynucleotide sequence.

In various embodiments, a method of using the apparatus and cartridgedescribed herein can further include one or more of the following steps:determining the presence of a polynucleotide sequence in the biologicalsample, the polynucleotide sequence corresponding to the probe, if theprobe is detected in the neutralized polynucleotide sample or a PCRamplicon thereof; determining that the sample was contaminated if theprobe is detected in the negative control polynucleotide or a PCRamplicon thereof; and/or in some embodiments, wherein the PCR reagentmixture further comprises a positive control plasmid and a plasmid probeselective for at least a portion of the plasmid, the method furtherincluding determining that a PCR amplification has occurred if theplasmid probe is detected.

Kit

In various embodiments, the microfluidic cartridge as described hereincan be provided in the form of a kit, wherein the kit can include amicrofluidic cartridge, and a liquid transfer member (such as a syringeor a pipette). In various embodiments, the kit can further includeinstructions to employ the liquid transfer member to transfer a samplecontaining extracted nucleic acid from a sample container via a sampleinlet to the microfluidic network on the microfluidic cartridge. In someembodiments, the microfluidic cartridge and the liquid transfer membercan be sealed in a pouch with an inert gas.

Typically when transferring a sample from liquid dispenser, such as apipette tip, to an inlet on the microfluidic cartridge, a volume of airis simultaneously introduced into the microfluidic network, the volumeof air being between about 0.5 mL and about 5 mL. Presence of air in themicrofluidic network, however, is not essential to operation of thecartridge described herein.

In various embodiments, the kit can further include at least onecomputer-readable label on the cartridge. The label can include, forexample, a bar code, a radio frequency tag or one or morecomputer-readable characters. When used in conjunction with a similarcomputer-readable label on a sample container, such as a vial or apouch, matching of diagnostic results with sample is therebyfacilitated.

In some embodiments, a sample identifier of the apparatus describedelsewhere herein is employed to read a label on the microfluidiccartridge and/or a label on the biological sample.

Heater Unit

An exemplary heater unit 2020 is shown in FIG. 26. The unit isconfigured to deliver localized heat to various selected regions of acartridge received in a receiving bay 2014. Heater unit 2020 isconfigured to be disposed within a diagnostic apparatus duringoperation, as further described herein, and in certain embodiments isremovable from that apparatus, for example to facilitate cleaning, or topermit reconfiguration of the heater circuitry. In various embodiments,heater unit 2020 can be specific to particular designs of microfluidicnetworks and microfluidic substrate layouts.

Shown in FIG. 26 is a heater unit having a recessed surface 2044 thatprovides a platform for supporting a microfluidic cartridge when inreceiving bay 2014. In one embodiment, the cartridge rests directly onsurface 2044. Surface 2044 is shown as recessed, in FIG. 2, but need notbe so and, for example, may be raised or may be flush with thesurrounding area of the heater unit. Surface 2044 is typically a layerof material that overlies a heater chip or board, or a heater substrate,that contains heater micro-circuitry configured to selectively andspecifically heat regions of a microfluidic substrate, such as in acartridge, in the receiving bay 2014.

Area 2044 is configured to accept a microfluidic cartridge in a singleorientation. Therefore area 2044 can be equipped with a registrationmember such as a mechanical key that prevents a user from placing acartridge into receiving bay 2014 in the wrong configuration. Shown inFIG. 26 as an exemplary mechanical key 2045 is a diagonally cutoutcorner of area 2044 into which a complementarily cutoff corner of amicrofluidic cartridge fits. Other registration members are consistentwith the heater unit described herein, for example, a feature engineeredon one or more edges of a cartridge including but not limited to:several, such as two or more, cut-out corners, one or more notches cutinto one or more edges of the cartridge; or one or more protrusionsfabricated into one or more edges of the cartridge. Alternativeregistration members include one or more lugs or bumps engineered intoan underside of a cartridge, complementary to one or more recessedsockets or holes in surface 2044 (not shown in the embodiment of FIG.26). Alternative registration members include one or more recessedsockets or holes engineered into an underside of a cartridge,complementary to one or more lugs or bumps on surface 2044. In general,the pattern of features is such that the cartridge possesses at leastone element of asymmetry so that it can only be inserted in a singleorientation into the receiving bay.

Also shown in FIG. 26 is a hand-grasp 2042 that facilitates removal andinsertion of the heater unit into an apparatus by a user. Cutaway 2048permits a user to easily remove a cartridge from receiving bay 2014after a processing run where, e.g., a user's thumb or finger whengrabbing the top of the cartridge, is afforded comfort space by cutaway2048. Both cutaways 2042 and 2048 are shown as semicircular recesses inthe embodiment of FIG. 26, but it would be understood that they are notso limited in shape. Thus, rectangular, square, triangular, half-oval,contoured, and other shaped recesses are also consistent with a heaterunit as described herein.

In the embodiment of FIG. 26, which is designed to be compatible with anexemplary apparatus as further described herein, the front of the heaterunit is at the left of the figure. At the rear of heater unit 2020 is anelectrical connection 2050, such as an RS-232 connection, that permitselectrical signals to be directed to heaters located at specific regionsof area 2044 during sample processing and analysis, as further describedherein. Thus, underneath area 2044 and not shown in FIG. 2 can be anarray of heat sources, such as resistive heaters, that are configured toalign with specified locations of a microfluidic cartridge properlyinserted into the receiving bay. Surface 2044 is able to be cleanedperiodically, for example with common cleaning agents (e.g., a 10%bleach solution), to ensure that any liquid spills that may occur duringsample handling do not cause any short circuiting. Such cleaning can becarried out frequently when the heater unit is disposed in a diagnosticapparatus, and less frequently but more thoroughly when the unit isremoved.

Other non-essential features of heater unit 2020 are as follows. One ormore air vents 2052 can be situated on one or more sides (such as front,rear, or flanking) or faces (such as top or bottom) of heater unit 2020,to permit excess heat to escape, when heaters underneath receiving bay2014, are in operation. The configuration of air vents in FIG. 26, as alinear array of square vents, is exemplary and it would be understoodthat other numbers and shapes thereof are consistent with routinefabrication and use of a heater unit. For example, although 5 square airvents are shown, other numbers such as 1, 2, 3, 4, 6, 8, or 10 air ventsare possible, arranged on one side, or spread over two or more sidesand/or faces of the heater unit. In further embodiments, air vents maybe circular, rectangular, oval, triangular, polygonal, and having curvedor squared vertices, or still other shapes, including irregular shapes.In further embodiments two or more vents need not be disposed in a line,parallel with one another and with an edge of the heater unit but may bedisposed offset from one another.

Heater unit 2020 may further comprise one or more guiding members 2047that facilitate inserting the heater unit into an apparatus as furtherdescribed herein, for an embodiment in which heater unit 2020 isremovable by a user. Heater unit is advantageously removable because itpermits system 2000 to be easily reconfigured for a different type ofanalysis, such as employing a different cartridge with a differentregistration member and/or microfluidic network, in conjunction with thesame or a different sequence of processing operations. In otherembodiments, heater unit 2020 is designed to be fixed and onlyremovable, e.g., for cleaning, replacement, or maintenance, by themanufacturer or an authorized maintenance agent, and not routinely bythe user. Guiding members 2047 may perform one or more roles of ensuringthat the heater unit is aligned correctly in the apparatus, and ensuringthat the heater unit makes a tight fit and does not significantly moveduring processing and analysis of a sample, or during transport of theapparatus.

Guiding members shown in the embodiment of FIG. 26 are on either side ofreceiving bay 2044 and stretch along a substantial fraction of thelength of unit 2020, but such an arrangement of guiding members isexemplary. Other guiding members are consistent with use herein, andinclude but are not limited to other numbers of guiding members such as1, 3, 4, 5, 6, or 8, and other positions thereof, including positionedin area 2051 of unit 2020, and need not stretch along as much of thelength of unit 2020 as shown in FIG. 26, or may stretch along its entirelength. Guiding members 2047 are shown having a non-constant thicknessalong their lengths. It is consistent herein that other guiding membersmay have essentially constant thickness along their lengths. At the endof the heater unit that is inserted into an apparatus, in the embodimentshown, the edges are beveled to facilitate proper placement.

Also shown in FIG. 26 is an optional region of fluorescent material,such as optically fluorescent material 2069, on area 2051 of heater unit2020. The region of fluorescent material is configured to be detected bya detection system further described herein. The region 2069 is used forverifying the state of optics in the detection system prior to sampleprocessing and analysis and therefore acts as a control, or a standard.For example, in one embodiment a lid of the apparatus in which theheater unit is disposed, when in an open position, permits ambient lightto reach region 2069 and thereby cause the fluorescent material to emita characteristic frequency or spectrum of light that can be measured bythe detector for, e.g., standardization or calibration purposes. Inanother embodiment, instead of relying on ambient light to cause thefluorescent material to fluoresce, light source from the detectionsystem itself, such as one or more LED's, is used to shine on region2069. The region 2069 is therefore positioned to align with a positionof a detector. Region 2069 is shown as rectangular, but may beconfigured in other shapes such as square, circular, elliptical,triangular, polygonal, and having curved or squared vertices. It is alsoto be understood that the region 2069 may be situated at other places onthe heater unit 2020, according to convenience and in order to becomplementary to the detection system deployed.

In particular and not shown in FIG. 26, heater/sensor unit 2020 caninclude, for example, a multiplexing function in a discrete multiplexingcircuit board (MUX board), one or more heaters (e.g., a microheater),one or more temperature sensors (optionally combined together as asingle heater/sensor unit with one or more respective microheaters,e.g., as photolithographically fabricated on fused silica substrates).The micro-heaters can provide thermal energy that can actuate variousmicrofluidic components on a suitably positioned microfluidic cartridge.A sensor (e.g., as a resistive temperature detector (RTD)) can enablereal time monitoring of the micro-heaters, for example through afeedback based mechanism to allow for rapid and accurate control of thetemperature. One or more microheaters can be aligned with correspondingmicrofluidic components (e.g., valves, pumps, gates, reaction chambers)to be heated on a suitably positioned microfluidic cartridge. Amicroheater can be designed to be slightly bigger than the correspondingmicrofluidic component(s) on the microfluidic cartridge so that eventhough the cartridge may be slightly misaligned, such as off-centered,from the heater, the individual components can be heated effectively.

Heater Configurations to Ensure Uniform Heating of a Region

The microfluidic substrates described herein are configured to acceptheat from a contact heat source, such as found in a heater unitdescribed herein. The heater unit typically comprises a heater board orheater chip that is configured to deliver heat to specific regions ofthe microfluidic substrate, including but not limited to one or moremicrofluidic components, at specific times. For example, the heat sourceis configured so that particular heating elements are situated adjacentto specific components of the microfluidic network on the substrate. Incertain embodiments, the apparatus uniformly controls the heating of aregion of a microfluidic network. In an exemplary embodiment, multipleheaters can be configured to simultaneously and uniformly heat a region,such as the PCR reaction chamber, of the microfluidic substrate. Theterm heater unit, as used herein, may be used interchangeably todescribe either the heater board or an item such as shown in FIG. 26.

Referring to FIGS. 27A and 27B, an exemplary set of heaters configuredto heat, cyclically, PCR reaction chamber 1001 is shown. It is to beunderstood that heater configurations to actuate other regions of amicrofluidic cartridge such as other gates, valves, and actuators, maybe designed and deployed according to similar principles to thosegoverning the heaters shown in FIGS. 27A and 27B.

Referring to FIGS. 27A and 27B, an exemplary PCR reaction chamber 1001in a microfluidic substrate, typically a chamber or channel having avolume ˜1.6 is configured with a long side and a short side, each withan associated heating element. A PCR reaction chamber may also bereferred to as a PCR reactor, herein, and the region of a cartridge inwhich the reaction chamber is situated may be called a zone. The heatersubstrate therefore includes four heaters disposed along the sides of,and configured to heat, a given PCR reaction chamber, as shown in theexemplary embodiment of FIG. 27A: long top heater 1005, long bottomheater 1003, short left heater 1007, and short right heater 1009. Thesmall gap between long top heater 1005 and long bottom heater 1003results in a negligible temperature gradient (less than 1° C. differenceacross the width of the PCR channel at any point along the length of thePCR reaction chamber) and therefore an effectively uniform temperaturethroughout the PCR reaction chamber. The heaters on the short edges ofthe PCR reactor provide heat to counteract the gradient created by thetwo long heaters from the center of the reactor to the edge of thereactor.

It would be understood by one of ordinary skill in the art that stillother configurations of one or more heater(s) situated about a PCRreaction chamber are consistent with the methods and apparatus describedherein. For example, a ‘long’ side of the reaction chamber can beconfigured to be heated by two or more heaters. Specific orientationsand configurations of heaters are used to create uniform zones ofheating even on substrates having poor thermal conductivity because thepoor thermal conductivity of glass, or quartz, polyimide, FR4, ceramic,or fused silica substrates is utilized to help in the independentoperation of various microfluidic components such as valves andindependent operation of the various PCR lanes. It would be furtherunderstood by one of ordinary skill in the art, that the principlesunderlying the configuration of heaters around a PCR reaction chamberare similarly applicable to the arrangement of heaters adjacent to othercomponents of the microfluidic cartridge, such as actuators, valves, andgates.

Generally, the heating of microfluidic components, such as a PCRreaction chamber, is controlled by passing currents through suitablyconfigured microfabricated heaters. Under control of suitable circuitry,the lanes of a multi-lane cartridge can then be controlled independentlyof one another. This can lead to a greater energy efficiency of theapparatus, because not all heaters are heating at the same time, and agiven heater is receiving current for only that fraction of the timewhen it is required to heat. Control systems and methods of controllablyheating various heating elements are further described in U.S. patentapplication Ser. No. 11/940,315, filed Nov. 14, 2007 and entitled“Heater Unit for Microfluidic Diagnostic System”.

In certain embodiments, each heater has an associated temperaturesensor. In the embodiment of FIG. 27A, a single temperature sensor 1011is used for both long heaters. A temperature sensor 1013 for short leftheater, and a temperature sensor 1015 for short right heater are alsoshown. The temperature sensor in the middle of the reactor is used toprovide feedback and control the amount of power supplied to the twolong heaters, whereas each of the short heaters has a dedicatedtemperature sensor placed adjacent to it in order to control it. Asfurther described herein, temperature sensors are preferably configuredto transmit information about temperature in their vicinity to aprocessor in the apparatus at such times as the heaters are notreceiving current that causes them to heat. This can be achieved withappropriate control of current cycles.

In order to reduce the number of sensor or heater elements required tocontrol a PCR heater, the heaters may be used to sense as well as heat,and thereby obviate the need to have a separate dedicated sensor foreach heater. In another embodiment, each of the four heaters may bedesigned to have an appropriate wattage, and connect the four heaters inseries or in parallel to reduce the number ofelectronically-controllable elements from four to just one, therebyreducing the burden on the associated electronic circuitry.

FIG. 27B shows expanded views of heaters and temperature sensors used inconjunction with a PCR reaction chamber of FIG. 27A. Temperature sensors1001 and 1013 are designed to have a room temperature resistance ofapproximately 200-300 ohms. This value of resistance is determined bycontrolling the thickness of the metal layer deposited (e.g., a sandwichof 400 Å TiW/3,000 Å Au/400 Å TiW), and etching the winding metal lineto have a width of approximately 10-25 μm and 20-40 mm length. The useof metal in this layer gives it a temperature coefficient of resistivityof the order of 0.5-20° C./ohms, preferably in the range of 1.5-3°C./ohms. Measuring the resistance at higher temperatures enablesdetermination of the exact temperature of the location of these sensors.

The configuration for uniform heating, shown in FIG. 27A for a singlePCR reaction chamber, can also be applied to a multi-lane PCR cartridgein which multiple independent PCR reactions occur.

Each heater can be independently controlled by a processor and/orcontrol circuitry used in conjunction with the apparatus describedherein. FIG. 27C shows thermal images, from the top surface of amicrofluidic cartridge when heated by heaters configured as in FIGS. 27Aand 27B, when each heater in turn is activated, as follows: (A): LongTop only; (B) Long Bottom only; (C) Short Left only; (D) Short Rightonly; and (E) All Four Heaters on. Panel (F) shows a view of thereaction chamber and heaters on the same scale as the other image panelsin FIG. 27C. Also shown in the figure is a temperature bar.

The configuration for uniform heating, shown in FIG. 27A for a singlePCR reaction chamber, can be applied to a multi-lane PCR cartridge inwhich multiple independent PCR reactions occur. See, e.g., FIG. 28,which shows an array of heater elements suitable to heat a cartridgeherein.

Heater Multiplexing (Under Software Control)

Another aspect of the heater unit described herein, relates to a controlof heat within the system and its components. The method leads to agreater energy efficiency of the apparatus described herein, because notall heaters are heating at the same time, and a given heater isreceiving current for only part of the time.

Generally, the heating of microfluidic components, such as a PCRreaction chamber, is controlled by passing currents through suitablyconfigured microfabricated heaters. The heating can be furthercontrolled by periodically turning the current on and off with varyingpulse width modulation (PWM), wherein pulse width modulation refers tothe on-time/off-time ratio for the current. The current can be suppliedby connecting a microfabricated heater to a high voltage source (forexample, 30 V), which can be gated by the PWM signal. In someembodiments, the device includes 48 PWM signal generators. Operation ofa PWM generator includes generating a signal with a chosen,programmable, period (the end count) and a particular granularity. Forinstance, the signal can be 4000 μs (micro-seconds) with a granularityof 1 μs, in which case the PWM generator can maintain a counterbeginning at zero and advancing in increments of 1 μs until it reaches4000 μs, when it returns to zero. Thus, the amount of heat produced canbe adjusted by adjusting the end count. A high end count corresponds toa greater length of time during which the microfabricated heaterreceives current and therefore a greater amount of heat produced. Itwould be understood that the granularity and signal width can takevalues other than those provided here without departing from theprinciples described herein.

Fluorescence Detection System, Including Lenses and Filters, andMultiple Parallel Detection for a Multi-Lane Cartridge

The detection system herein is configured to monitor fluorescence comingfrom one or more species involved in a biochemical reaction. The systemcan be, for example, an optical detector having a light source thatselectively emits light in an absorption band of a fluorescent dye, anda light detector that selectively detects light in an emission band ofthe fluorescent dye, wherein the fluorescent dye corresponds to afluorescent polynucleotide probe or a fragment thereof, as furtherdescribed elsewhere herein. Alternatively, the optical detector caninclude a bandpass-filtered diode that selectively emits light in theabsorption band of the fluorescent dye and a bandpass filteredphotodiode that selectively detects light in the emission band of thefluorescent dye. For example, the optical detector can be configured toindependently detect a plurality of fluorescent dyes having differentfluorescent emission spectra, wherein each fluorescent dye correspondsto a fluorescent polynucleotide probe or a fragment thereof. Forexample, the optical detector can be configured to independently detecta plurality of fluorescent dyes at a plurality of different locationsof, for example, a microfluidic substrate, wherein each fluorescent dyecorresponds to a fluorescent polynucleotide probe or a fragment thereof.The detector further has potential for 2, 3 or 4 color detection and iscontrolled by software, preferably custom software, configured to sampleinformation from the detector.

The detection system described herein is capable of detecting afluorescence signal from nanoliter scale PCR reactions. Advantageously,the detector is formed from inexpensive components, having no movingparts. The detector can be configured to couple to a microfluidiccartridge as further described herein, and can also be part of apressure application system, such as a sliding lid on an apparatus inwhich the detector is situated, that keeps the cartridge in place.

FIGS. 29-31B depict an embodiment of a highly sensitive fluorescencedetection system that includes light emitting diodes (LED's),photodiodes, and filters/lenses for monitoring, in real-time, one ormore fluorescent signals emanating from the microfluidic channel. Theembodiment in FIGS. 29-31B displays a two-color detection system havinga modular design that couples with a single microfluidic channel found,for example, in a microfluidic cartridge. It would be understood by oneskilled in the art that the description herein could also be adapted tocreate a detector that just detects a single color of light. FIGS. 31Aand 31B show elements of optical detector elements 1220 including lightsources 1232 (for example, light emitting diodes), lenses 1234, lightdetectors 1236 (for example, photodiodes) and filters 1238. The detectorcomprises two LED's (blue and red, respectively) and two photodiodes.The two LED's are configured to transmit a beam of focused light on to aparticular region of the cartridge. The two photo diodes are configuredto receive light that is emitted from the region of the cartridge. Onephotodiode is configured to detect emitted red light, and the otherphotodiode is configured to detect emitted blue light. Thus, in thisembodiment, two colors can be detected simultaneously from a singlelocation. Such a detection system can be configured to receive lightfrom multiple microfluidic channels by being mounted on an assembly thatpermits it to slide over and across the multiple channels. The filterscan be, for example, bandpass filters, the filters at the light sourcescorresponding to the absorption band of one or more fluorogenic probesand the filters at the detectors corresponding to the emission band ofthe fluorogenic probes.

FIGS. 32 and 33 show an exemplary read-head comprising a multiplexed 2color detection system that is configured to mate with a multi-lanemicrofluidic cartridge. FIG. 32 shows a view of the exterior of amultiplexed read-head. FIG. 33 is an exploded view that shows howvarious detectors are configured within an exemplary multiplexed readhead, and in communication with an electronic circuit board.

Each of the detection systems multiplexed in the assembly of FIGS. 32and 33 is similar in construction to the embodiment of FIGS. 29-31B. Themodule in FIGS. 32 and 33 is configured to detect fluorescence from eachof 12 microfluidic channels, as found in, for example, the respectivelanes of a 12-lane microfluidic cartridge. Such a module thereforecomprises 24 independently controllable detectors, arranged as 12 pairsof identical detection elements. Each pair of elements is then capableof dual-color detection of a pre-determined set of fluorescent probes.It would be understood by one of ordinary skill in the art that othernumbers of pairs of detectors are consistent with the apparatusdescribed herein. For example, 4, 6, 8, 10, 16, 20, 24, 25, 30, 32, 36,40, and 48 pairs are also consistent and can be configured according tomethods and criteria understood by one of ordinary skill in the art.

Detection Sensitivity, Time Constant and Gain

A typical circuit that includes a detector as described herein includes,in series, a preamplifier, a buffer/inverter, a filter, and a digitizer.Sensitivity is important so that high gain is very desirable. In oneembodiment of the preamplifier, a very large, for example 100 GΩ,resistor is placed in parallel with the diode. Other values of aresistor would be consistent with the technology herein: such valuestypically fall in the range 0.5-100 GΩ, such as 1-50 GΩ, or 2-10 GΩ. Anexemplary pre-amplifier circuit configured in this way is shown in FIG.7. Symbols in the figure have their standard meanings in electroniccircuit diagrams.

The FIG. 34 shows a current-to-voltage converter/pre-amplifier circuitsuitable for use with the detection system. D11 is the photodetectorthat collects the fluorescent light coming from the microfluidic channeland converts it into an electric current. The accompanying circuitry isused to convert these fluorescent currents into voltages suitable formeasurement and output as a final measure of the fluorescence.

A resistor-capacitor circuit in FIG. 34 contains capacitor C45 andresistor R25. Together, the values of capacitance of C45 and resistanceof R25 are chosen so as to impact the time constant τ_(c) (equal to theproduct of R25 and C45) of the circuit as well as gain of the detectioncircuit. The higher the time constant, the more sluggish is the responseof the system to incident light. It typically takes the duration of afew time constants for the photodetector to completely charge to itsmaximum current or to discharge to zero from its saturation value. It isimportant for the photo current to decay to zero between measurements,however. As the PCR systems described herein are intended to affordrapid detection measurements, the product R₂₅C₄₅ should therefore bemade as low as possible. However, the gain of the pre-amplifier whosecircuitry is shown is directly proportional to the(fluorescent-activated) current generated in the photodetector and theresistance R₂₅. As the fluorescence signal from the microfluidic channeldevice is very faint (due to low liquid volume as well as small pathlengths of excitation), it is thus important to maximize the value ofR₂₅. In some embodiments, R₂₅ is as high as 100 Giga-Ohms (for example,in the range 10-100 GΩ), effectively behaving as an open-circuit. Withsuch values, the time-constant can take on a value of approximately50-100 ms by using a low-value capacitor for C45. For example, thelowest possible available standard off-the-shelf capacitor has a valueof 1 pF (1 picoFarad). A time-constant in the range 50-100 ms ensuresthat the photocurrent decays to zero in approximately 0.5 s (approx. 6cycles) and therefore that approximately 2 samplings can be made persecond. Other time constants are consistent with effective use of thetechnology herein, such as in the range 10 ms-1 s, or in the range 50ms-500 ms, or in the range 100-200 ms. The actual time constant suitablefor a given application will vary according to circumstance and choiceof capacitor and resistor values. Additionally, the gain achieved by thepre-amplifier circuit herein may be in the range of 10⁷-5×10⁹, forexample may be 1×10⁹.

As the resistance value for R25 is very high (˜100 GΩ), the manner ofassembly of this resistor on the optics board is important for theoverall efficiency of the circuit. Effective cleaning of the circuitduring assembly and before use is important to achieve an optimaltime-constant and gain for the optics circuit.

It is also important to test each photo-diode that is used, because manydo not perform according to specification.

Sensitivity and Aperturing

The LED light passes through a filter before passing through the samplein the microfluidic channel (as described herein, typically 300 μldeep). This is a very small optical path-length for the light in thesample. The generated fluorescence then also goes through a secondfilter, and into a photo-detector. Ultimately, then, the detector mustbe capable of detecting very little fluorescence. Various aspects of thedetector configuration can improve sensitivity, however.

The illumination optics can be designed so that the excitation lightfalling on the PCR reactor is incident along an area that is similar tothe shape of the reactor. As the reactor is typically long and narrow,the illumination spot should be long and narrow, i.e., extended, aswell. The length of the spot can be adjusted by altering a number offactors, including: the diameter of the bore where the LED is placed(the tube that holds the filter and lens has an aperturing effect); thedistance of the LED from the PCR reactor; and the use of proper lens atthe right distance in between. As the width of the beam incident on thereactor is determined by the bore of the optical element (approximately6 mm in diameter), it is typical to use an aperture (a slit having awidth approximately equal to the width of the reactor, and a lengthequal to the length of the detection volume) to make an optimalillumination spot. A typical spot, then, is commensurate with thedimensions of a PCR reaction chamber, for example 1.5 mm wide by 7 mmlong. FIG. 35A shows the illumination spot across 12 PCR reactors forthe two different colors used. A typical aperture is made of anodizedaluminum and has very low autofluoresence in the wavelengths ofinterest. FIG. 35B illustrates a cross-section of a detector, showing anexemplary location for an aperture 802.

The optimal spot size and intensity is importantly dependent on theability to maintain the correct position of the LED's with respect tothe center of the optical axis. Special alignment procedures and checkscan be utilized to optimize this. The different illuminations can alsobe normalized with respect to each other by adjusting the power currentthrough each of the LED's or adjusting the fluorescence collection time(the duration for which the photodetector is on before measuring thevoltage) for each detection spot. It is also important to align thedetectors with the axis of the micro-channels.

The aperturing is also important for successful fluorescence detectionbecause as the cross-sectional area of the incident beam increases insize, so the background fluorescence increases, and the fluorescenceattributable only to the molecules of interest (PCR probes) gets masked.Thus, as the beam area increases, the use of an aperture increases theproportion of collected fluorescence that originates only from the PCRreactor. Note that the aperture used in the detector herein not onlyhelps collect fluorescence only from the reaction volume but itcorrespondingly adjusts the excitation light to mostly excite thereaction volume. The excitation and emission aperture is, of course, thesame.

Based on a typical geometry of the optical exctiation and emissionsystem and aperturing, show spot sizes as small as 0.5 mm by 0.5 mm andas long as 8 mm×1.5 mm have been found to be effective. By using a longdetector (having an active area 6 mm by 1 mm) and proper lensing, theaperture design can extend the detection spot to as long as 15-20 mm,while maintaining a width of 1-2 mm using an aperture. Correspondingly,the background fluorescence decreases as the spot size is decreased,thereby increasing the detection sensitivity.

Use of Detection System to Measure/Detect Fluid in PCR Chamber

The fluorescence detector is sensitive enough to be able to collectfluorescence light from a PCR chamber of a microfluidic substrate. Thedetector can also be used to detect the presence of liquid in thechamber, a measurement that provides a determination of whether or notto carry out a PCR cycle for that chamber. For example, in amulti-sample cartridge, not all chambers will have been loaded withsample; for those that are not, it would be unnecessary to apply aheating protocol thereto. One way to determine presence or absence of aliquid is as follows. A background reading is taken prior to filling thechamber with liquid. Another reading is taken after microfluidicoperations have been performed that should result in filling the PCRchamber with liquid. The presence of liquid alters the fluorescencereading from the chamber. A programmable threshold value can be used totune an algorithm programmed into a processor that controls operation ofthe apparatus as further described herein (for example, the secondreading has to exceed the first reading by 20%). If the two readings donot differ beyond the programmed margin, the liquid is deemed to nothave entered the chamber, and a PCR cycle is not initiated for thatchamber. Instead, a warning is issued to a user.

Exemplary Electronics and Software

The heater unit described herein can be controlled by variouselectronics circuitry, itself operating on receipt ofcomputer-controlled instructions. FIG. 36 illustrates exemplaryelectronics architecture modules for operating a heater unit anddiagnostic apparatus. It would be understood by one of ordinary skill inthe art that other configurations of electronics components areconsistent with operation of the apparatus as described herein. In theexemplary embodiment, the electronics architecture is distributed acrosstwo components of the apparatus: the Analyzer 2100 and a Heater unit2102. The Analyzer apparatus as further described herein contains, forexample, an Optical Detection Unit 2108, a Control Board 2114, aBackplane 2112, and a LCD Touchscreen 2110. The Control Board includes aCard Engine 2116 further described herein, and Compact Flash memory2118, as well as other components. The Heater Assembly includes a HeaterBoard 2104 and a Heater Mux Board 2106, both further described herein.

In one embodiment, the Card Engine electronics module 2116 is acommercial, off the shelf “single board computer” containing aprocessor, memory, and flash memory for operating system storage.

The optional LCD+Touchscreen electronics module 2110 is a userinterface, for example, driven through a touchscreen, such as a 640pixel by 480 pixel 8 inch LCD and 5-wire touchscreen.

The Compact Flash electronics module 2118 is, for example, a 256megabyte commercial, off the shelf, compact flash module for applicationand data storage. Other media are alternatively usable, such asUSB-drive, smart media card, memory stick, and smart data-card havingthe same or other storage capacities.

The Backplane electronics module 2112 is a point of connection for theremovable heater assembly 2102. Bare PC boards with two connectors aresufficient to provide the necessary level of connectivity.

The Control Board electronics module 2114 supports peripherals to theCard Engine electronics module 2116. In one embodiment, the peripheralsinclude such devices as a USB host+slave or hub, a USB CDROM interface,serial ports, and ethernet ports. The Control Board 2114 can include apower monitor with a dedicated processor. The Control Board may alsoinclude a real time clock. The Control Board may further include aspeaker. The Control Board 2114 also includes a CPLD to provide SPIaccess to all other modules and programming access to all other modules.The Control Board includes a programmable high voltage power supply. TheControl Board includes a Serial-Deserializer interface to theLCD+Touchscreen electronics module 2110 and to the Optical DetectionUnit electronics module 2108. The Control Board also includes moduleconnectors.

In the exemplary embodiment, the optical detection unit electronicsmodule 2108 contains a dedicated processor. The optical detection unit2108 contains a serializer-deserializer interface. The optical detectionunit 2108 contains LED drivers. The optical detection unit also containshigh gain-low noise photodiode amplifiers. The optical detection unitcan have power monitoring capability. The optical detection unit canalso be remotely reprogrammable.

The Heater Board electronics module 2104 is preferably a glass heaterboard. The Heater Board has PCB with bonding pads for glass heater boardand high density connectors.

In one embodiment, the heater mux (‘multiplex’) board electronics module2106 has 24 high-speed ADC, 24 precision current sources, and 96optically isolated current drivers for heating. The heater mux board hasthe ability to time-multiplex heating/measurement. The heater mux boardhas multiplexer banks to multiplex inputs to ADC, and to multiplexcurrent source outputs. The heater mux board has a FPGA with a softprocessor core and SDRAM. The heater mux board has a Power Monitor witha dedicated processor. The Heater Mux Board can be remotelyreprogrammable.

In another embodiment, control electronics can be spread over fourdifferent circuit board assemblies. These include the MAIN board: Canserve as the hub of the Analyzer control electronics and managescommunication and control of the other various electronic subassemblies.The main board can also serve as the electrical and communicationsinterface with the external world. An external power supply (12V DC/10A; UL certified) can be used to power the system. The unit cancommunicate via 5 USB ports, a serial port and an Ethernet port.Finally, the main board can incorporate several diagnostic/safetyfeatures to ensure safe and robust operation of the Analyzer.

MUX Board: Upon instruction from the main board, the MUX board canperform all the functions typically used for accurate temperaturecontrol of the heaters and can coordinate the collection of fluorescencedata from the detector board.

LCD Board: Can contain the typical control elements to light up the LCDpanel and interpret the signals from the touch sensitive screen. TheLCD/touch screen combination can serve as a mode of interaction with theuser via a Graphical User Interface.

Detector Board: Can house typical control and processing circuitry thatcan be employed to collect, digitize, filter, and transmit the data fromthe fluorescence detection modules.

Certain software can be executed in each electronics module. The ControlBoard Electronics Module executes, for example, Control Board PowerMonitor software. The Card Engine electronics module executes anoperating system, graphical user interface (GUI) software, an analyzermodule, and an application program interface (api). The OpticalDetection Unit electronics module executes an optics software module.The Heater Mux Board electronics module executes dedicated Heater Muxsoftware, and Heater Mux Power Monitor software. Each of the separateinstances of software can be modular and under a unified control of, forexample, driver software.

The exemplary electronics can use Linux, UNIX, Windows, or MacOS,including any version thereof, as the operating system. The operatingsystem is preferably loaded with drivers for USB, Ethernet, LCD,touchscreen, and removable media devices such as compact flash.Miscellaneous programs for configuring the Ethernet interface, managingUSB connections, and updating via CD-ROM can also be included.

In the embodiment of FIG. 36, the analyzer module is the driver forspecific hardware. The analyzer module provides access to the Heater MuxModule, the Optical Detection Unit, the Control Board Power Monitor, theReal Time Clock, the High Voltage Power Supply, and the LCD backlight.The analyzer module provides firmware programming access to the ControlBoard power monitor, the Optical Detection Unit, and the Heater MuxModule.

The API provides uniform access to the analyzer module driver. The APIis responsible for error trapping, and interrupt handling. The API istypically programmed to be thread safe.

The GUI software can be based on a commercial, off-the-shelf PEGgraphics library. The GUI can use the API to coordinate the self-test ofoptical detection unit and heater assembly. The GUI starts, stops, andmonitors test progress. The GUI can also implement an algorithm toarrive on diagnosis from fluorescence data. The GUI provides accesscontrol to unit and in some embodiments has an HIS/LIS interface.

The Control Board Power Monitor software monitors power supplies,current and voltage, and signals error in case of a fault.

The Optics Software performs fluorescence detection which is preciselytimed to turn on/off of LED with synchronous digitization of thephotodetector outputs. The Optics Software can also monitor power supplyvoltages. The Optics Software can also have self test ability.

The Heater Mux Module software implements a “protocol player” whichexecutes series of defined “steps” where each “step” can turn on sets ofheaters to implement a desired microfluidic action. The Heater MuxModule software also has self test ability. The Heater Mux Modulesoftware contains a fuzzy logic temperature control algorithm.

The Heater Mux Power Monitor software monitors voltage and currentlevels. The Heater Mux Power Monitor software can participate inself-test, synchronous, monitoring of the current levels while turningon different heaters.

EXAMPLES

The following are exemplary aspects of various parts and functions ofthe system described herein.

Additional embodiments of a cartridge are found in U.S. patentapplication Ser. No. 11/940,310, entitled “Microfluidic Cartridge andMethod of Making Same”, and filed on even date herewith, thespecification of which is incorporated herein by reference.

Additional embodiments of heater units and arrays are described in U.S.patent application Ser. No. 11/940,315, entitled “Heater Unit forMicrofluidic Diagnostic System” and filed on even date herewith, thespecification of which is incorporated herein by reference in itsentirety.

Further description of suitably configured detectors are described inU.S. patent application Ser. No. 11/940,321, filed on Nov. 14, 2007 andentitled “Fluorescence Detector for Microfluidic Diagnostic System”,incorporated herein by reference.

Example 1: Analyzer Having Removable Heater Unit

This non-limiting example describes pictorially, various embodiments ofan apparatus, showing incorporation of a heater unit and a microfluidiccartridge operated on by the heater unit.

FIG. 37 shows an apparatus 1100 that includes a housing having a displayoutput 1102, an openable lid 1104, and a bar code reader 1106. Thecartridge is positioned in a single orientation in a receiving bay underthe lid, FIG. 38. The lid of the apparatus can be closed to applypressure to the cartridge, as shown in FIG. 39. The unit currentlyweighs about 20 lbs. and is approximately 10″ wide by 16″ deep by 13″high.

FIGS. 40 and 41: The heating stage of the apparatus can be removable forcleaning, maintenance, or to replace a custom heating stage for aparticular microfluidic cartridge. FIGS. 40 and 41 also show how aheater unit is insertable and removable from a front access door to ananalyzer apparatus.

Example 2: Assembly of an Exemplary Heater Unit

FIG. 42A shows an exploded view of an exemplary heater unit. The unithas a top cover and a bottom cover that together enclose a Mux board(control board), a pressure support layer, and insulator film, and amicrothermal circuit on a PCB. The last of these is the heat source thatselectively heats regions of a microfluidic substrate placed in contacttherewith through the top cover.

An exemplary heater substrate, FIG. 42B, consists of aphoto-lithographically processed glass wafer bonded to a standard 0.100″standard FR4 printed circuit board. The glass wafer is 0.5 mm thick andis cut into a rectangle the size of ˜3.5×4.25 inches. The glasssubstrate has numerous metal heaters and resistive temperature sensorsphoto-lithographically etched on the surface of the glass wafer. Thesubstrate is aligned and bonded to the PCBoard using a compliant epoxy,ensuring flatness to within 2-3 mils over the surface of the wafer. Thecured epoxy should withstand up to 120° C. for two hours minimum.Approximately 300-400 bond pads of the size of approximately 1 mm×0.25mm, with exposed gold surfaces, are located along the two long edges ofthe wafer. These pads are wirebonded (ball-bonding) to correspondingpads on the PCB using 1.5 mil gold wires. Wire bonding is a threadingprocess, standard in semiconductor FAB. Alternatively, a flip-chipmethod may be used, though such methods are more complicated and maywarp the wafer because of thermal mismatch. Wire bonds should have goodintegrity and pass defined pull strength. The substrate is baked at 120°C. for two hours and then the wire bonds are encapsulated by a compliantepoxy that will protect the wirebonds but not damage the bonds even at120° C. Encapsulant should not spill over predefined area around thewirebonds and should not be taller than a defined height. For example,instead of laying epoxy all over the substrate, lines (e.g., a hashpattern) of it are made so that epoxy cures and air escapes throughside. Alternatively, a laminate fill (adhesive on both sides) can beused. Standard connectors are soldered to the PCB and then the unit istested using a test set-up to ensure all heaters and sensors read theright resistance values.

Pictures of an exemplary Mux board and assembled heater unit are shownin FIGS. 27-29.

Example 3: Pulse Width Modulation for Heater Circuitry

In various embodiments, the operation of a PWM generator can alsoinclude a programmable start count in addition to the aforementioned endcount and granularity. In such embodiments, multiple PWM generators canproduce signals that can be selectively non-overlapping (e.g., bymultiplexing the on-time of the various heaters) such that the currentcapacity of the high voltage power is not exceeded. Multiple heaters canbe controlled by different PWM signal generators with varying start andend counts. The heaters can be divided into banks, whereby a bankdefines a group of heaters of the same start count. For example, 36 PWMgenerators can be grouped into six different banks, each correspondingto a certain portion of the PWM cycle (500 ms for this example). The endcount for each PWM generator can be selectively programmed such that notmore than six heaters will be on at any given time. A portion of a PWMcycle can be selected as dead time (count 3000 to 4000 for this example)during which no heating takes place and sensitive temperature sensingcircuits can use this time to sense the temperature. The table belowrepresents a PWM cycle for the foregoing example:

Start Count End Count Max End count Bank 1 PWM generator#1   0  150  500PWM generator#2   0  220  500 . . . . . . . . . . . . PWM generator#6  0  376  500 Bank 2 PWM generator#7  500  704 1000 PWM generator#8  500 676 1000 . . . . . . . . . . . . PWM generator#12  500  780 1000 Bank 3PWM generator#13 1000 1240 1500 PWM generator#14 1000 1101 1500 . . . .. . . . . . . . PWM generator#18 1000 1409 1500 Bank 4 PWM generator#191500 1679 2000 PWM generator#20 1500 1989 2000 . . . . . . . . . . . .PWM generator#24 1500 1502 2000 Bank 5 PWM generator#25 2000 2090 2500PWM generator#26 2000 2499 2500 . . . . . . . . . . . . PWM generator#302000 2301 2500 Bank 6 PWM generator#31 2500 2569 3000 PWM generator#322500 2790 3000 . . . . . . . . . . . . PWM generator#36 2500 2678 3000

Example 4: Detector Integrated in Force Member

This non-limiting example describes pictorially, various embodiments ofa detection system integrated into a force member, in an apparatus forcarrying out diagnostics on microfluidic samples.

FIG. 43A: The lid of the apparatus can be closed, which can blockambient light from the sample bay, and place an optical detectorcontained in the lid into position with respect to the microfluidiccartridge.

FIG. 43B: The lid of the apparatus can be closed to apply pressure tothe cartridge. Application of minimal pressure on the cartridge: afterthe slider compresses the cartridge, the slider can compress thecompliant label of the cartridge. This can cause the bottom of thecartridge to be pressed down against the surface of the heater unitpresent in the heater module. Springs present in the slider can deliver,for example approximately 50 lb of pressure to generate a minimumpressure, for example 2 psi over the entire cartridge bottom.

Thermal interface: the cartridge bottom can have a layer of mechanicallycompliant heat transfer laminate that can enable thermal contact betweenthe microfluidic substrate and the microheater substrate of the heatermodule. A minimal pressure of 1 psi can be employed for reliableoperation of the thermal valves, gate and pumps present in themicrofluidic cartridge.

Mechanicals and assembly: the Analyzer can have a simple mechanicalframe to hold the various modules in alignment. The optics module can beplaced in rails for easy opening and placement of cartridges in theAnalyzer and error-free alignment of the optics upon closing. Theheater/sensor module can be also placed on rails or similar guidingmembers for easy removal and insertion of the assembly.

Slider: the slider of the Analyzer can house the optical detectionsystem as well as the mechanical assembly that can enables the opticsjig to press down on the cartridge when the handle of the slider isturned down onto the analyzer. The optics jig can be suspended from thecase of the slider at 4 points. Upon closing the slider and turning thehandle of the analyzer down, 4 cams can turn to push down a plate thatpresses on 4 springs. On compression, the springs can deliverapproximately 50 lb on the optical block. See FIGS. 44A-44C.

The bottom surface of the optics block can be made flat to within 100microns, typically within 25 microns, and this flat surface can pressupon the compliant (shore hardness approximately 50-70) label(approximately 1.5 mm thick under no compression) of the cartridgemaking the pressure more or less uniform over the cartridge. An optionallock-in mechanism can also be incorporated to prevent the slider frombeing accidentally knocked-off while in use.

FIG. 45A shows a side view of a lever assembly 1200, with lever 1210,gear unit 1212, and force member 1214. Assembly 1200 can be used toclose the lid of the apparatus and (through force members 1214) applyforce to a microfluidic cartridge 1216 in the receiving chamber 1217.One force member is visible in this cut away view, but any number, forexample 4, can be used. The force members can be, for example, a manualspring loaded actuator as shown, an automatic mechanical actuator, amaterial with sufficient mechanical compliance and stiffness (e.g., ahard elastomeric plug), and the like. The force applied to themicrofluidic cartridge 1216 can result in a pressure at the surface ofthe microfluidic cartridge 1216 of at least about 0.7 psi to about 7 psi(between about 5 and about 50 kilopascals), or in some embodiments about2 psi (about 14 kilopascals.

FIG. 45B shows a side view of lever assembly 1200, with microfluidiccartridge 1216 in the receiving chamber 1217. A heat source 1219 (forexample, a xenon bulb as shown) can function as a radiant heat sourcedirected at a sample inlet reservoir 1218, where the heat can lyse cellsin reservoir 1218. A thermally conductive, mechanically compliant layer1222 can lie at an interface between microfluidic cartridge 1216 andthermal stage 1224. Typically, microfluidic cartridge 1216 and thermalstage 1224 can be planar at their respective interface surfaces, e.g.,planar within about 100 microns, or more typically within about 25microns. Layer 1222 can improve thermal coupling between microfluidiccartridge 1216 and thermal stage 1224. Optical detector elements 1220can be directed at the top surface of microfluidic cartridge 1216.

FIGS. 45C and 45D show further cross-sectional views.

Example 6: Exemplary Optics Board

An exemplary optics board is shown schematically in FIG. 46, and is usedto collect and amplify the fluorescent signature of a successfulchemical reaction on a micro-fluidic cartridge, and control theintensity of LED's using pulse-width modulation (PWM) to illuminate thecartridge sample over up to four channels, each with two color options.Additionally, it receives instructions and sends results data back overan LVDS (low-voltage differential signaling) SPI (serial peripheralinterface). In some embodiments there is a separate instance of thiscircuitry for each PCR channel that is monitored.

The power board systems include: a +12V input; and +3.3V, +3.6V, +5V,and −5V outputs, configured as follows: the +3.3V output contains alinear regulator, is used to power the LVDS interface, should maintain a+/−5% accuracy, and supply an output current of 0.35 A; the +3.6V outputcontains a linear regulator, is used to power the MSP430, shouldmaintain a +/−5% accuracy, and supply an output current of 0.35 A; the+5V output contains a linear regulator, is used to power the plus railfor op-amps, should maintain a +/−5% accuracy, and supply an outputcurrent of 0.35 A; the −5V output receives its power from the +5Vsupply, has a mV reference, is used to power the minus rail for op-ampsand for the photo-detector bias, should maintain a +/−1% voltageaccuracy, and supply an output current of 6.25 mA+/−10%. Additionally,the power board has an 80 ohm source resistance, and the main boardsoftware can enable/disable the regulator outputs.

The main board interface uses a single channel of the LVDS standard tocommunicate between boards. This takes place using SPI signaling overthe LVDS interface which is connected to the main SPI port of thecontrol processor. The interface also contains a serial port forin-system programming.

The optical detection system of FIG. 46 comprises a control processor,LED drivers, and a photo-detection system. In the exemplary embodiment,the control processor is a TI MSP430F1611 consisting of a dual SPI (onefor main board interface, and one for ADC interface) and extended SRAMfor data storage. It has the functions of power monitoring, PWM LEDcontrol, and SPI linking to the ADC and main board. The LED driverscontain NPN transistor switches, are connected to the PWM outputs of thecontrol processor, can sink 10 mA @ 12V per LED (80 mA total), and aresingle channel with 2 LEDs (one of each color) connected to each. Thephoto-detection system has two channels and consists of aphoto-detector, high-sensitivity photo-diode detector, high gain currentto voltage converter, unity gain voltage inverting amplifier, and anADC. Additionally it contains a 16 channel Sigma-delta (only utilizingthe first 8 channels) which is connected to the second SPI port of thecontrol processor.

During assembly of the various components on to the PC board, such asmay occur on a production line, there are the following considerations.The extremely high impedance of the photo-detection circuit means that arigorous cleaning procedure must be employed. Such a procedure mayinclude, for example: After surface mount components are installed, theboards are washed on a Weskleen and blow dried upon exiting conveyor.The belt speed can be set at 20-30. The boards are soaked in an alcoholbath for approximately 3 minutes, then their entire top and bottomsurfaces are scrubbed using a clean, soft bristle brush. The boards arebaked in a 105° F. (40° C.) oven for 30 minutes to dry out allcomponents.

After all the components are installed: the soldered areas of the boardscan be hand wash using deionized water and a soft bristle brush. Thesame soldered areas can be hand washed using alcohol and a soft bristlebrush. The boards are allowed to air dry. Once the board is cleaned, theoptical circuitry must be conformal coated to keep contaminates out.

The foregoing description is intended to illustrate various aspects ofthe present technology. It is not intended that the examples presentedherein limit the scope of the present technology. The technology nowbeing fully described, it will be apparent to one of ordinary skill inthe art that many changes and modifications can be made thereto withoutdeparting from the spirit or scope of the appended claims.

The invention claimed is:
 1. A fully automated, continuous random-accessmolecular diagnostic test system comprising: a plurality of consumablescomprising: a plurality of multi-sample microfluidic cartridges, eachmicrofluidic cartridge comprising a plurality of separate sample inlets,each microfluidic cartridge configured to receive a plurality ofseparate amplification-ready nucleic acid-containing samples at theplurality of separate sample inlets, each nucleic acid-containing sampleassociated with a microfluidic network of a plurality of microfluidicnetworks, each microfluidic network comprising an amplification regionwithin which nucleic acid amplification is carried out when thenucleic-acid containing sample is received in the microfluidiccartridge, and a plurality of disposable pipette tips; a plurality ofaccessories comprising: a test strip carrier, and a microfluidiccartridge carrier; and an instrument comprising a touchscreen computer,a handheld barcode scanner, a keyboard, a mouse, and a power supply, theinstrument further comprising: a receiving bay configured to receive amicrofluidic cartridge of the plurality of multi-sample microfluidiccartridges, a liquid-handling robot comprising a plurality of dispensingheads configured to transfer nucleic acid-containing samples to theplurality of separate sample inlets when the microfluidic cartridge isreceived in the receiving bay, a plurality of separately controllableheat sources configured at the receiving bay, each heat sourceconfigured to thermal cycle a nucleic acid-containing sample in anamplification region of the microfluidic cartridge when received in thereceiving bay, wherein the amplification region is maintained at asubstantially uniform temperature at least once in each thermal cycle,one or more processors configured to control the plurality of separatelycontrollable heat sources when the microfluidic cartridge is received inthe receiving bay, and an optical scanner configured to measure a levelof fluorescence emitted from each amplification region when themicrofluidic cartridge is received in the receiving bay, the opticalscanner further configured to provide real-time detection of anamplified target nucleic acid sequence in the respective amplificationregion, wherein the touchscreen computer is configured to display a testresult for a nucleic-acid containing sample amplified in themicrofluidic cartridge based at least in part on the level offluorescence measured by the optical scanner.
 2. The system of claim 1,wherein the system is configured for automated extraction and isolationof nucleic acids from multiple specimen types for a sample to resulttime of about one hour, with test results released continuously afterthe first test result.
 3. The system of claim 2, wherein the opticalscanner is configured to measure fluorescence at multiple wavelengthsthereby enabling multiplexed detection in each amplification region inthe microfluidic cartridge when received in the receiving bay.
 4. Thesystem of claim 3, wherein the plurality of separate sample inlets arealigned along a first axis of the microfluidic cartridge, wherein theamplification regions are aligned along a second axis substantiallyparallel to the first axis, and wherein the optical scanner does notcover the plurality of separate sample inlets when the microfluidiccartridge is received in the receiving bay.
 5. The system of claim 4,wherein, when the microfluidic cartridge is received in the receivingbay, the liquid-handling robot is configured to transfer a nucleicacid-containing sample to the microfluidic cartridge after a differentnucleic acid-containing sample has been amplified in the microfluidiccartridge.
 6. The system of claim 5, wherein the liquid handling robotis configured to transfer a first plurality of nucleic acid-containingsamples to a first set of separate sample inlets of the microfluidiccartridge when received in the receiving bay, and wherein the liquidhandling robot is further configured to subsequently transfer a secondplurality of nucleic acid-containing samples to a second set of separatesample inlets of the microfluidic cartridge after the first plurality ofnucleic acid-containing samples has been amplified in the microfluidiccartridge.
 7. The system of claim 4, further comprising a secondreceiving bay configured to receive a second microfluidic cartridge ofthe plurality of multi-sample microfluidic cartridges, wherein thesystem is configured to: concurrently process a first nucleicacid-containing sample in the microfluidic cartridge when received inthe receiving bay and a second nucleic acid-containing sample in thesecond microfluidic cartridge when received in the second receiving bay;consecutively process a third nucleic acid-containing sample in themicrofluidic cartridge when received in the receiving bay and a fourthnucleic acid-containing sample in the second microfluidic cartridge whenreceived in the second receiving bay; and display a test result for thefirst, second, third, and fourth nucleic acid-containing samples on thetouchscreen computer.
 8. The system of claim 7, wherein the touchscreencomputer is configured to display a test result for a nucleic-acidcontaining sample amplified in an amplification region of themicrofluidic cartridge based at least in part on a level of fluorescenceemitted from the amplification region by an amplified control nucleicacid sequence.
 9. The system of claim 8, wherein the amplified targetnucleic acid sequence is from an organism selected from the groupconsisting of: bacteria, a virus, human immunodeficiency virus, humanpapilloma virus, hepatitis B virus, hepatitis C virus, influenza virus,Group B Streptococcus, Group A Streptococcus, Chlamydia trachomatis,Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium.10. The system of claim 9, wherein the system is configured to performamplification and detection on 18 or more than 18 nucleicacid-containing samples in about an hour.
 11. The system of claim 10,wherein, prior to performing real-time detection, the system isconfigured to perform a fill check to ensure that an amplificationregion is filled with a nucleic acid-containing sample when themicrofluidic cartridge is received in the receiving bay and thenucleic-acid containing sample is transferred to the microfluidiccartridge.
 12. The system of claim 1, wherein the optical scanner isconfigured to measure fluorescence at multiple wavelengths therebyenabling multiplexed detection in each amplification region in themicrofluidic cartridge when received in the receiving bay.
 13. Thesystem of claim 1, wherein the plurality of separate sample inlets arealigned along a first axis of the microfluidic cartridge, wherein theamplification regions are aligned along a second axis substantiallyparallel to the first axis, and wherein the optical scanner does notcover the plurality of separate sample inlets when the microfluidiccartridge is received in the receiving bay.
 14. The system of claim 1,wherein, when the microfluidic cartridge is received in the receivingbay, the liquid-handling robot is configured to transfer a nucleicacid-containing sample to the microfluidic cartridge after a differentnucleic acid-containing sample has been amplified in the microfluidiccartridge.
 15. The system of claim 1, wherein the liquid handling robotis configured to transfer a first plurality of nucleic acid-containingsamples to a first set of separate sample inlets of the microfluidiccartridge when received in the receiving bay, and wherein the liquidhandling robot is further configured to subsequently transfer a secondplurality of nucleic acid-containing samples to a second set of separatesample inlets of the microfluidic cartridge after the first plurality ofnucleic acid-containing samples has been amplified in the microfluidiccartridge.
 16. The system of claim 1, further comprising a secondreceiving bay configured to receive a second microfluidic cartridge ofthe plurality of multi-sample microfluidic cartridges, wherein thesystem is configured to: concurrently process a first nucleicacid-containing sample in the microfluidic cartridge when received inthe receiving bay and a second nucleic acid-containing sample in thesecond microfluidic cartridge when received in the second receiving bay;consecutively process a third nucleic acid-containing sample in themicrofluidic cartridge when received in the receiving bay and a fourthnucleic acid-containing sample in the second microfluidic cartridge whenreceived in the second receiving bay; and display a test result for thefirst, second, third, and fourth nucleic acid-containing samples on thetouchscreen computer.
 17. The system of claim 1, wherein the touchscreencomputer is configured to display a test result for a nucleic-acidcontaining sample amplified in an amplification region of themicrofluidic cartridge based at least in part on a level of fluorescenceemitted from the amplification region by an amplified control nucleicacid sequence.
 18. The system of claim 1, wherein the amplified targetnucleic acid sequence is from an organism selected from the groupconsisting of: bacteria, a virus, human immunodeficiency virus, humanpapilloma virus, hepatitis B virus, hepatitis C virus, influenza virus,Group B Streptococcus, Group A Streptococcus, Chlamydia trachomatis,Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium.19. The system of claim 1, wherein the system is configured to performamplification and detection on 18 or more than 18 nucleicacid-containing samples in about an hour.
 20. The system of claim 1,wherein, prior to performing real-time detection, the system isconfigured to perform a fill check to ensure that an amplificationregion is filled with a nucleic acid-containing sample when themicrofluidic cartridge is received in the receiving bay and thenucleic-acid containing sample is transferred to the microfluidiccartridge.